Class JR-SiP) H 

Book, 5 n 

CoKTtghtN? 



A 



LABORATORY SYLLABUS 



OF 



CLINICAL PATHOLOGY 



BY 

CHARLES E. SIMON, B.A., M.D. 

PROFESSOR OF CLINICAL PATHOLOGY IN THE SCHOOL OF MEDICINE AND THE COLLEGE OF 
PHYSICLANS AND SURGEONS OF THE UNIVERSITY OF MARYLAND , BALTIMORE, MD. 




LEA & FEBIGER 

PHILADELPHIA AND NEW YORK 
1919 



Copyright 
LEA & FEBIGER 
1919 



NOV 15 1919 



©CI.A53659 9 



PREFACE. 



Every teacher of clinical pathology, no doubt, has observed, 
with alarm, the rapid growth in volume of the subject-matter 
which he is obliged to take up in his classes, while the number of 
hours that are placed at his disposal for this purpose is remaining 
practically the same. Not many years ago the course in clinical 
microscopy, as it was then and is still at times wrongly called, 
was largely a course in what the name then suggested, viz., a 
course in the microscopic examination of the blood and the various 
secretions and excretions of the body, coupled with a relatively 
meager review of some of the simpler methods of chemical analysis 
of the same material. 

Nowadays a course along these older lines would no longer be 
deemed sufficient to familiarize the student with those methods 
of laboratory investigation which are in daily use in any up-to- 
date clinical laboratory, and a knowledge of which is rightly 
regarded as essential by a board of modern medical examiners. 

At the University of Maryland the class in clinical pathology 
is divided into two sections, the instruction of each of which 
extends over a period of fifty-eight afternoons, of two hours each, 
with thirteen additional single-hour periods. This covers the 
time spent in the laboratory proper, in lectures, demonstrations 
and recitations. Our principal difficulty in the past has been 
to provide adequate time for actual laboratory work and reci- 
tations. During the laboratory periods a certain amount of 
time has, in the past, been spent in explaining the work to be 
done, thus leaving not more than one hour and a half for actual 
work. The recitation work, of course, has suffered the most, 
and gradually has become a negligible factor; as a consequence 
systematic home reading is practically not done at all. 

Various measures have suggested themselves as remedies for 
this undesirable state of affairs, the simplest evidently being an 



iv 



PREFACE 



increase in the number of teaching hours. But until a general 
readjustment of the curriculum, of the third and fourth years 
more particularly, is adopted by all the medical schools of our 
country, this course is as yet not available. 

Meanwhile, it has occurred to me, that a considerable amount 
of time might be gained by eliminating the brief talks at the 
beginning of each lesson, which after all do not cover the ground 
as fully as is actually desirable, and by substituting therefor a set 
of printed instructions which would serve as a guide to the after- 
noon's work. With this idea in mind I have written this little 
syllabus. It will be noted that the subject-matter has been 
arranged in connection with each lesson under three headings, 
viz.: (1) instructions to the assistants regarding the nature of 
the material, the reagents and apparatus that are required for 
each lesson; (2) instructions to the student for the afternoon's 
work, with directions as to the manner in which his findings and 
results are to be reported; (3) a set of questions based on the work 
done in the laboratory and upon home reading, which are to be 
answered in writing at home and the answers presented to the 
instructor as a part of the work done. 

The plan then is, from time to time, to call in the books in 
which these data are assembled, for criticism and correction. 
This involves a good deal of work on the part of the instructor, 
to be sure, which cannot be avoided, however, and will serve to 
keep him in touch with the status and the progress made by the 
individual student, which, after all, is as it should be. 

It will be noted that the subject-matter to be covered in the 
laboratory has been arranged on the basis of thirty-nine lessons, 
each lesson to occupy a two-hour period. This I am inclined to 
regard as the minimum of time that should be available for actual 
practical work. Where more time is at the instructor's disposal 
some of the lessons may, of course, be advantageously repeated, 
but I dare say that the majority of teachers will not be able to 
devote many more hours to work in the laboratory proper, as they 
will require at least eighteen hours for lectures and as many for 
recitations. The table of contents will give an idea of the work 
that is to be covered in the laboratory. 

In conclusion, I would draw attention to the heading "Read- 



PREFACE 



V 



ing" at the end of each lesson. I have left this blank so thai 
any instructor who does not use my own text-book in his course 
may give the page references to his students in those works which 
happen to be his favorites. 

Trusting that this little book may be found serviceable by the 
student and the instructor alike, and that the latter by its use 
may gain more time for his lectures and demonstrations than 
was at his command heretofore, I submit it to both with the hope 
that it may be accorded the same generous reception that my 
Clinical Diagnosis has met with throughout its many editions. 

Charles E. Simon. 

1734 Linden Avenue, 
Baltimore, Md. 



CONTENTS. 



Lesson 1. — Study of the morphological elements of the blood in the 

unstained specimen 17 

Lesson 2. — The enumeration of the red cells and the leukocytes of the 

blood 18 

Lesson 3. — The estimation of the hemoglobin (a) according to Talquist; 

(b) according to Sahli; (c) according to v. Fleischl 19 

Lesson 4. — The behavior of the morphological elements of the blood 
toward anilin dyes, viz., (a) toward an acid dye; (6) toward a basic 
dye; (c) toward a neutral staining mixture (a) Jenner's stain, (jS) 

Wright's stain 20 

Lesson 5. — (a) The differential leukocyte count under normal conditions. 

(b) The leukocytic formula in typhoid fever 23 

Lesson 6. — The leukocytic formula in pyogenic and tubercular infections 25 
Lesson 7. — The leukocytic formula in whooping-cough, intestinal parasitic 

infections, in chronic malaria 26 

Lesson 8. — The leukocytic formula in acute lymphatic leukemia, in 

chronic lymphatic leukemia and chronic myelocytic leukemia . . 20 
Lesson 9. — The morphological blood picture in chlorosis, in the secondary 

anemias and in pernicious anemia 27 

Lesson 10. — The agglutination reaction in typhoid and paratyphoid 
fever: (a) the macroscopic slide agglutination method; (6) the micro- 
scopic slide agglutination method; (c) the macroscopic test-tube 

method 29 

Lesson 11. — The examination of a supposed fecal typhoid carrier . . . 31 

Lesson 12. — Continuation of Lesson 11 32 

Lesson 13. — The Wassermann reaction; general technic 34 

Lesson 14. — The Wassermann reaction; titration of amboceptor and 

antigen 37 

Lesson 15. — The transfusion test 40 

Lesson 16. — The estimation of the blood sugar: (a) in a normal indi- 
vidual; (b) in a diabetic 42 

Lesson 17. — The estimation of the blood urea 43 

Lesson 18. — The microscopic examination of transudates and exudates . 46 
Lesson 19. — The examination of sputum for tubercle bacilli .... 47 
Lesson 20. — The examination of pneumonic sputum and the type deter- 
mination of pneumococci 49 

Lesson 21. — Continuation of Lesson 20 51 

Lesson 22. — The bacteriological study of the flora of the throat and naso- 
pharynx; the examination of supposed carriers of the diphtheria 
bacillus, of the Streptococcus viridans, the Streptococcus hcmolyticus 
and the meningococcus 53 



CONTENTS vii 

Lesson 23. — Continuation of Lesson 22 55 

Lesson 24. — Continuation of Lesson 23 50 

Lesson 25. — The examination of the cerebrospinal fluid : (a) The Was 
mann test; (b) Lange's colloidal gold reaction; (c) the Ross-Jones 

test; (d) the cell count 57 

Lesson 26. — The cytological study of the cerebrospinal fluid under 

various pathological conditions 59 

Lesson 27. — The chemical examination of the stomach contents; quanti- 
tative determination of the various acid fractions; tests for lactic 

acid; test for pepsin and chymosin 60 

Lesson 28. — Microscopical examination of the stomach contents and 
feces, with the exception of the parasitology of the latter; tests for 

occult blood 62 

Lesson 29. — The malarial parasite 64 

Lesson 30. — Trypanosomes and Leishmania; disease-carrying insects 66 

Lesson 31. — Ameba histolytica and Ameba coli 66 

Lesson 32. — Trichomonas, Lamblia and Balantidium 68 

Lesson 33. — The ova of Taenia solium, Taenia saginata,, Hymenolepis 
nana, Dibothriocephalus latus, Fasciola hepatica, Clonorchis endemi- 
cus, Paragonimus Westermani, Schistosomum hematobium and 

Schistosomum japonicum 70 

Lesson 34. — The adult parasites corresponding to the ova considered in 
Lesson 33; the ova of Ascaris lumbricoides, Oxyuris vermicularis, 

Necator americanus and Trichocephalus trichiurus 71 

Lesson 35. — The adult parasites corresponding to the ova considered in 
Lesson 34; Trichinella spiralis; Strongyloides intestinalis ; tissue speci- 
mens showing hookworms and trichinella cysts in situ . . . 73 
Lesson 36. — The routine chemical examination of the urine .... 75 
Lesson 37. — The quantitative estimation of albumin, of sugar and the 

chlorides in urine 78 

Lesson 38. — The permeation test; the study of acidosis by the deter- 
mination of the alveolar carbon dioxide tension 80 

Lesson 39. — The microscopical examination of the urine 83 



LABORATORY SYLLABUS OF CLINICAL 
PATHOLOGY. 



Lesson 1. — Apparatus and Reagents Required. — Slides and cover- 
glasses (No. 1) that have been cleansed with soap and watt i ; 
sterilized gauze sponges in pairs, wrapped in muslin; Hagedorn 
needles; individual bottles of alcohol (2 oz.). 

Cleanse the lobe of the ear of your working partner with alcohol; 
dry; puncture with needle that has previously been placed in alcohol 
for a couple of minutes and dried; wipe away first drop; mount next 
drop (small size) on a cover-glass and invert immediately on a siide; 
the drop should spread out by capillary attraction; if this does not 
occur, make another mount. Examine with oil-immersion lens. 

Note and draw appearances of red cells where they are well 
separated. 

Answer the Following Questions in Writing. — What is the form and 
color of the normal red cell? what is its size? what are the normal 
variations in size ? what does the central pale area denote ? what is 
meant by the following terms: normocyte, microcyte, macrocyte, 
microcytosis, macrocytosis, anisocytosis, poikilocyte, poikilocytosis. 

Note and draw crenated red cells; cells in money- roll formation. 
Note and draw the various types of leukocytes that occur in normal 
blood, with the exception of the so-called mast cell, viz., the small 
mononuclear, the large mononuclear, the finely granular poly- 
morphonuclear and the coarsely granular polymorphonuclear, 
indicating size and form of nucleus, the relation in bulk between 
nucleus and cytoplasm and the presence or absence ami si i 
of granules. 

Draw leukocytes undergoing ameboid movements 
Answer the Following Questions in Writing. — What is the size [a) 
of a small, and of (b) a large mononuclear leukocyte cell, (c of a 
finely granular and (c/) of a coarsely granular polymorphonuclear 
leukocyte as compared with a red cell. What type is the most 
common? what next in order? which next and which last? which 
are capable of undergoing ameboid movements? which are capable 



18 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



of phagocytic activity? which type after emigration from the 
bloodvessels constitutes the common pus corpuscle? 
Reading. — 

Lesson 2. — Apparatus and Reagents Required. — Hemocytometers ; 
normal salt solution, 3 per cent, acetic acid and alcohol in individual 
bottles (2 oz.); Hagedorn needles; sterilized gauze sponges in pairs, 
wrapped in muslin. 

Examine and draw (a) the red pipette, permitting a dilution of 
1 to 100 and 1 to 200; (b) the white pipette, giving a dilution of 1 
to 10 and 1 to 20, and (c) the counting slide — the latter in profile 
with the cover-glass adjusted over the central ruled area. Examine 
the ruling with the low power (objective 3 L. or | B. & L.) and 
draw it. 

Answer the Following Questions in Writing. — What diluting fluid 
do you use (a) when making red counts? (b) when making white 
counts? What degree of dilution do you commonly use (a) when 
making red counts? (6) when making white counts? why? 

Using Simon's counting chamber, what is the size of a small 
square? what of a rectangle? what of a large square? what are the 
corresponding cubic contents of a mounted drop ? In which squares 
do you count the red cells and in which the white? 

Experiment . — (a) Puncture the ear of your working partner, as 
described in Lesson 1 ; draw blood into your red pipette to the mark 
0.5 and saline to the mark 101, giving a dilution of 1 to 200 in the 
bulb; detach the rubber tubing and shake for several minutes; blow 
out the diluting fluid in the stem of the instrument and mount a 
drop of the contents of the bulb on your counting surface; adjust 
the cover-glass; slide this to and fro until Newton's colored rings 
appear; let stand for a few minutes and now count the red cells in 
100 small squares, using the low power of the microscope, marking 
them down in the diagram, that you have drawn, with a colored 
pencil; those on the top and left lines are counted in and those on 
the right and bottom lines out. Determine the average per one 
small square and multiply by the corresponding cubic contents and 
the degree of the dilution. The resultant figure indicates the number 
of red cells in 1 c.mm. of blood. 

(b) Charge the white pipette in a similar manner to the mark 
0.5; dilute to the mark 11 with 3 per cent, acetic acid and shake for 
several minutes; blow out the diluting fluid from the stem of the 
instrument; mount a drop of the diluted blood on the counting 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY L9 

surface; adjust the cover-glass; let stand for a few minutes, then 
count the leukocytes contained in 100 of the large squares, using 
first the middle power of the microscope and repeating with the low 
power, marking down the cells in the diagram that you have drawn. 
Determine the average for a single large square; multiply by the 
corresponding cubic contents and the degree of dilution. The 
resultant figure indicates the number of leukocytes in 1 c.mm. of 
blood. 

Answer the Following Questions in Writing. — Why does the mixture 
of blood and acetic acid in the bulb of your mixing pipette turn 
brown ? what is formed ? 

What is the normal number of (a) the red cells, and (b) of the 
leukocytes per c.mm. of blood. What is meant by the following 
terms: oligocythemia, polycythemia, polyglobulism? What is the 
difference between relative and absolute polycythemia? Name con- 
ditions under which the former and the latter may be encountered. 
How high and how low may the red count go in disease. What is 
meant by the following terms: leukocytosis, hypoleukocytosis, 
leukopenia, hyperleukocytosis. How high and how low may the 
leukocyte count go in disease? 

Reading. — 

Lesson 3. — Apparatus and Reagents Required. — Talquist's color 
scale; Sahli's hemoglobinometer; v. Fleischl's hemometer; medicine 
droppers; distilled water; normal salt solution; decinormal hydro- 
chloric acid and alcohol in individual bottles (2 oz.); Hagedora 
needles; sterilized gauze sponges in pairs wrapped in muslin. 

1. Make hemoglobin estimations according to the three methods 
indicated and compare results, using the blood of your working 
partner, (a) Using Talquist's color scale, mount a drop of blood 
on a piece of the accompanying filter paper and compare the color 
while still wet with the color scale of the Talquist booklet; note the 
result. 

(b) In using Sahli's hemoglobinometer, place a little decinormal 
hydrochloric acid in the graduated test-tube, say up to the 20 mark. 
Fill the blood pipette up to the 20 c.mm. mark with blood, in solid 
column, and gently blow this out into the decinormal acid; -hake 
until the diluted blood has turned brown and is perfectly transparent ; 
add water, a couple of drops at a time, and shake after each addition 
until the color of the mixture when placed in the accompanying 
little stand, alongside the standard solution, matches that of the 



20 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

latter; the figure corresponding to the level reached indicates the 
percentage of hemoglobin. 

(c) Using the v. Fleischl instrument, half-fill each chamber of the 
well that fits the central aperture of the stage of the instrument 
with water. Puncture the ear or finger and allow one of the capil- 
lary pipettes that accompany the apparatus to fill itself by merely 
holding one end in contact with the drop. Transfer this volume 
of blood to one of the chambers in which water has just been placed 
and agitate the pipette gently until all blood has diffused out. 
Wash through with a few drops of water; fill both chambers with 
water until a convex meniscus is formed at the top of each. Adjust 
the colored wedge under the stage of the instrument; turn the well 
so that the half, filled with water only, stands above the wedge; 
by means of the rack-and-pinion adjustment, turn the latter to and 
fro until the colors in the two chambers match; this must be done 
in a darkened room, using artificial light; the colors are best com- 
pared by viewing them through a paper tube, so as to exclude all 
outside light. The figure opposite the little notch in the scale well, 
on the stage of the instrument, indicates the percentage of hemo- 
globin. 

2. Do a red count in the same person whose hemoglobin has just 
been studied, as described in Lesson 2. Determine the percentage 
of the normal that the count represents, taking 5,000,000 as average 
normal, and hence as representing 100 per cent. 

3. Determine the color index for the individual examined by 
dividing the percentage of hemoglobin by the percentage of red cells. 

Answer the Following Questions in Writing. — What is the normal 
percentage of hemoglobin, gravimetrically expressed? What rela- 
tion exists between a 100 per cent, reading obtained by means of a 
hemoglobinometer and the actual amount of hemoglobin ? To what 
percentage, gravimetrically, would a reading of say 65 per cent, 
correspond ? 

What is meant by the term color index? what is it normally? 
what does an increased index usually indicate? what is the index 
in chlorosis? what in the secondary anemias? What is meant by 
oligochromemia? hypocytochromia ? hypercytochromia ? What is 
the supposed explanation of the latter? 

Reading. — 

Lesson 4. — Apparatus and Reagents Required. — Clean slides; 
Hagedorn needles; gauze sponges in pairs, wrapped in muslin; 
alcohol; (a) a 1 per cent, aqueous solution of eosin; (6) a 1 per cent. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 21 

aqueous solution of methylene blue; Jenner's stain; Wright's stain 
— all reagents in individual bottles; staining trays; distilled water 
in 100 c.c. lots for every six men; tumblers or wash bottles for 
washing with tap water; blotting or filter paper for individual use. 

Cleanse needle and lobe of the ear with alcohol; puncture; mount 
a moderate-sized drop of blood near the end of one slide; bring the 
narrow edge of a second slide in contact with the blood and let the 
drop spread along the edge by capillary attraction; then, without 
using any pressure, sweep the edge of the second slide along the 
entire surface of the first, starting at an acute angle, rapidly coming 
to a right angle and ending at an obtuse angle. The resultant 
smear will appear in ridges. Make half a dozen smears and let dry 
in the air. These will keep in good condition for staining for at least 
a week when wrapped in paper. Place two smears on the bars of 
the staining tray; cover with alcohol to fix and leave for five minutes; 
wash off with tap water; cover one with eosin solution (acid dye) 
and the other with methylene-blue solution (basic dye); stain for 
one minute; wash off; allow to dry in the air or blot with filter 
paper. Stain a third specimen, without preliminary fixation, for 
three or four minutes with Jenner's stain (a neutral mixture of eosin 
and methylene blue), by flooding the slide with the stain; wash off 
with tap water and allow to dry in the air. Stain a fourth specimen 
with Wright's stain (a neutral mixture of eosin and methylene 
azure) as follows: Flood the slide on the bars of the staining tray 
with Wright's stain. After forty-five seconds add an equivalent 
volume of distilled water; leave the mixture for four minutes; wash 
off the stain with tap water and dry the specimen by blotting. 

Examine the eosin specimen first. Note (a) that the red cells 
have taken the eosin stain intensely, (6) that the nuclei of all types 
of leukocytes are scarcely stained at all, or at most a faint pinkish — 
action upon the achromatic substance of the nuclei; (c) that the 
cytoplasm of the small and large mononuclear leukocytes is likewise 
nearly colorless or a faint pink — action upon the paraplasmic com- 
ponent of the cytoplasm ; id) that the cytoplasm of the finely gran- 
ular polymorphonuclear leukocytes stains a more definite pinkish 
color while the granules are colorless; (e) that the granules of the 
coarsely granular polymorphonuclear leukocytes are stained a 
bright red. 

Material that stains with eosin is said to be eosinophilic or oxy- 
philic. 

Illustrate the above points, using red pencils, and note the degree 
of oxyphilia by the mark + ++> =*= and o. 
2 



22 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

Examine the methylene-blue specimen next: Note (a) that the 
nuclei of all the different types of leukocytes have taken the basic 
methylene-blue stain; they are hence said to be basophilic. The 
same is true of the cytoplasm — spongioplasm — of the small and large 
mononuclear leukocytes and to a slight extent of the spongioplasm 
of the eosinophilic — coarsely granular leukocytes. The granules of 
the finely and coarsely granular leukocytes remain colorless, but 
if by any chance a so-called mast-cell be encountered, it will be 
noted that its granules are deeply colored by the basic dye. The 
red cells hardly take the stain at all — the slight staining is prob- 
ably due to the presence of traces of the original spongioplasm of 
the cell. 

Illustrate the above points, using a blue pencil for all basophilic 
elements, and note the degree of basophilia by the marks + ++, 
++, + and 0. 

Next examine the Jenner — eosinate of methylene-blue — specimen. 
Note that the red cells take a reddish-brownish tone, due to the 
marked affinity for the acid stain on the part of the hemoglobin and 
the slight affinity for the basic stain on the part of the basophilic 
component of the cell. All cell nuclei are colored blue, as is also the 
cyto-spongioplasm of the small and large mononuclear leukocytes; 
to a less extent that of the eosinophiles, while the granules of the 
mast cells are deeply colored by the methylene-blue; the eosinophilic 
granules appear a bright red and the fine granules of the common 
polymorphonuclear leukocytes, which took neither the acid nor the 
basic stain, now are colored a fine purplish red ; they are hence said 
to be neutrophilic. The blood platelets which may be seen here and 
there among the red cells, or coming from their interior through a 
cleft, are colored light blue. 

Illustrate all these appearances, using colored pencils, and note 
which elements are oxyphilic, basophilic, and neutrophilic. 

Finally, examine the Wright specimen. Note the brilliant color 
of the cell nuclei — reddish purple — which is due to the methylene 
azure; in some of the large and small mononuclears isolated granules 
will be seen, colored reddish purple, the so-called azurophilic granules; 
the neutrophilic granules are not as well stained as in the Jenner 
specimen; the eosinophilic granules appear a brownish red, but are 
not as striking as in the Jenner preparation; the basophilic mast-cell 
granules appear a reddish purple; the cytoplasm of the small and 
large mononuclears is practically colorless; the blood-platelets 
appear a bluish gray, with a reddish-purple central portion. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY S.\ 



Illustrate the various appearances, using colored pencils, and note 
which elements are azurophilic. 

Answer the Following Questions in Writing. — Which chemical 
group in the make-up of the aniline dye determines whether the 
product has (a) acid or (b) basic properties? What is meant by a 
neutral dye mixture? Name an acid dye, a basic dye, two neutral 
mixtures? What is the salt-forming group of a dye called? Wliat 
is meant by the chromogenic group of a dye? Give an example 
of a chromogenic group. W T hat is meant by the terms oxyphilic, 
eosinophilic, neutrophilic, basophilic, azurophilic, orthochromatic, 
metachromatic. 

Which is the essential basic constituent of all the so-called Roman- 
owsky mixtures? name some of these mixtures. Wliat relationship 
exists between methylene-blue and methylene azure? What is a 
polychrome or panoptic stain ? 

Why is it unnecessary to fix with alcohol before using Jenner's 
or Wright's stain ? 

Wliat volumetric relation exists between nucleus and cytoplasm 
(a) in the small, (b) in the large mononuclear leukocyte? Does this 
rule hold for all cells of this type irrespective of their age? What is 
a "transition form," (a) morphologically considered, (b) in its 
relation to the large mononuclear leukocyte? 

W 7 hat is meant by a polynuclear cell, as contrasted with a poly- 
morphonuclear cell? 

What are the essential morphological points of difference between 
the neutrophilic and eosinophilic leukocytes of normal blood. 

Reading. — 

Lesson 5. — Apparatus and Reagents Required. — Clean slides; 
sterile gauze sponges, in pairs, covered with muslin; Hagedorn 
needles; alcohol; Jenners stain, Wright's stain in individual 
bottles (2 oz.); staining trays; distilled water; wash bottles; filter 
paper. 

Students should also be furnished with stained blood smears from 
(a) a typhoid case about the end of the first week; (b) a typhoid case 
from the third week; (c) a typhoid convalescent whose temperature 
has been normal for several days. 

1. Prepare blood films from your working partner and stain with 
Jenner's stain, as described in Lesson 4. Wipe the reverse side of the 
slide dry and examine while the blood side is still wet, using- the 
lowest power of the microscope. (Leitz obj. 3, ocular 4). Note (a) 
the size, the color and general distribution of the red cells; (b) the 



24 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



leukocytes among the red cells which appear as little blue dots; 
with practice the different types may readily be differentiated from 
one another; the small mononuclears appear as purely blue dots, 
the size of a red cell; the large mononuclears are a lighter blue and 
much larger than a red cell; the polynuclear neutrophils show a 
lobed blue nucleus lying in a slightly pinkish cell body; the eosino- 
phils show a lobed blue nucleus lying in a bright red cell body; in 
the basophiles the granules appear almost black, scattered over the 
surface of the cell body, while the nucleus appears light blue. 

Try to differentiate the various types in every blood smear that 
you examine until you feel as confident of your ability to do so with 
the low power as with the oil-immersion lens. While making your 
low-power survey, get in the habit of noting the number of leuko- 
cytes in a field. In the course of time you will learn to estimate 
their absolute number by simple inspection. If your slide becomes 
dry while working with the low power wet it again with water. After 
having finished your low-power survey, allow the slide to become 
air-dry and then examine with the oil-immersion lens. The best 
portion of the slide is the first third, counting from the point where 
the drop of blood was first mounted. Move the slide up and down, 
and, following the ridges of the smear, note in tabular form all the 
leukocytes that you encounter, in sets of five, as shown below; count 
an even 300 and calculate the percentages of the different types. 



Small mononuclears: TUl T!U Tffl 7Hi etc - • • 80 

Large mononuclears: ffH ff£l Jf£[ 15 

Polynuclear neutrophils : ffl-l 7HI 7M HAL > etc - • • 198 

Polynuclear eosinophils : j 6 

Polynuclear basophiles: / 1 



300 

Having familiarized yourself with the different types of cells, 
practice counting a large number, in your mind, before putting 
them down, and then note them as follows: 



S.M. 


L.M. P.M. 






P.E. 


P.B. 


12 


4 52 






1 


0 


26 


7 40 






2 


0 


20 


8 46 






1 


0 


20 


7 50 






2 


2 


78 


26 188 






6 


2 = 300 




Small mononuclears 


= 26.0 per cent. 






Large mononuclears 


= 8 


.6 








Polynuclear neutrophiles 


= 62 


.6 








Polynuclear eosinophiles 


= 2. 


0 








Polynuclear basophiles 


= 0. 


6 







LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 25 



A count of this order is termed a differential count. 

Now do a so-called Arneth count: To this end classify a hundred 
neutrophiles according to the number of separate nuclear lobes, 
arranging them in five groups. 

Study in the same manner the blood smears from a typhoid 
patient obtained (a) at the end of the first week; (b) in the course 
of the third week of the disease; (c) during the first week of con- 
valescence. Arrange your data in tabular form under the headings: 

(a) Differential count; (b) Arneth count. 

Answer the Following Questions in Writing. — What are the normal 
percentages of the different leukocytes (a) in the adult; (b) in chil- 
dren younger than six years? What is meant by lymphocytosis, 
splenocytosis, neutrophilic hyperleukocytosis, neutrophilic hypo- 
leukocytosis, eosinophilia, hypereosinophilia, hypoeosinophilia, 
basophilia? What is meant by Simon's septic factor? 

Name some pathological conditions (a) in which the neutro- 
philic elements are increased; (b) in which they are diminished; (c) 
in which the eosinophiles are increased; (d) in which they are 
diminished; (e) in which the small mononuclears are increased; (/) 
in which the large mononuclears are increased. 

Supposing the total count in a given case was 2500 and the differ- 
ential count showed 50 per cent, of small mononuclears and 40 per 
per cent, of polynuclear neutrophiles, which factor would you 
regard as the essential one — the decrease of the polys or the increase 
of the small monos; calculate the corresponding absolute values per 
cubic millimeter, and compare them with the normal. 

Reading. — 

Lesson 6. — Students to be furnished with stained blood smears 
from the following pathological conditions: (a) acute appendicitis, 

(b) pneumococcus pneumonia; (c) meningococcus meningitis; (</) 
streptococcus endocarditis; (e) an acute exacerbation of a pulmonary 
tuberculosis; (/) tubercular osteomyelitis. Use Jenner's solution 
for staining purposes. 

Examine the specimens first with the low-power and then with 
the oil-immersion lens. Note with the low-power whether the 
total number of the leukocytes appears to be increased, and the 
extent, as compared with a slide of normal blood; then do a dif- 
ferential count, paying particular attention to the presence oral 
of eosinophiles; compare the findings in the first four cases with 
those in the last two. Give your findings in the form of a formal 
report. 



26 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

Ansiver the Followiiig Questions in Writing. — In infections with 
what organisms do you find an increase of the neutrophils asso- 
ciated with a decrease or absence of eosinophils, and in which do 
you find an increase of the neutrophiles associated with normal or 
even increased percentage values of the eosinophiles? What do 
you mean by an epicritic eosinophilia? 

Lesson 7. — Students to be supplied with stained smears from 
(a) a case of whooping-cough; (b) a quiescent pulmonary tuber- 
culosis, (c) a case of tapeworm infection, (d) a case of chronic 
lymphatic leukemia, (e) a case of hookworm infection, (/) from a 
chronic malarial infection. Study these various slides as in Lesson 
6, (a) with a low power, noting the total number of the leukocytes 
as compared with the normal; (b) do a differential count in each. 
What are the salient features in each case? 

Reading. — 

Lesson 8. — Students to be supplied with blood smears (a) from a 
case of acute lymphatic leukemia, stained according to Wright's 
method; (b) from a case of chronic lymphatic leukemia, stained 
according to Wright; (c) from a case of chronic myelocytic leukemia, 
stained with Jenner's stain. 

With the low power note the total number of leukocytes, as com- 
pared with the normal, and gauge the extent of the increase in actual 
numbers. Examine the first smear with the oil-immersion and care- 
fully compare the structure of the predominating cell and its nucleus 
with that controlling the picture in the second case. Make drawings 
illustrating the size of these cells, as compared with the size of a red 
cell, and of the arrangement of the chromatin in the nucleus. The 
predominating cell in the first specimen is the so-called lymphoido- 
cyte; in addition cells will be seen which correspond to the descrip- 
tion of the leukoblast; search also for Rieder forms, i. e., cells of the 
types mentioned in which the nucleus has become polymorphous. 
Incidentally, note the pallor of the red cells and the occurrence 
of nucleated red cells — almost exclusively normoblasts. Make a 
differential count in both (a) and (6); while doing so note the 
number of nucleated red cells that you encounter while counting 
300 leukocytes. While differentiating the lymphoidocytes in speci- 
men (a) group them into two categories, viz., those which are dis- 
tinctly larger than a red cell, macrolymphoidocytes, and those which 
correspond to the small monos in size, microlymphoidocytes. Can 
you find any true small monos (lymphocytes) in the smear? Now 
examine the specimen from the case of chronic myelocytic leukemia. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 27 



Note the following points: (a) the enormous increase in the total 
number of the leukocytes — gauge it; (b) that nearly all the leuko- 
cytic elements are granule-bearing; (c) that many of the neutro- 
philic and eosinophilic polymorphs are smaller than normal; (ri) 
that many neutrophilic and eosinophilic elements are mononuclear — 
myelocytes; (e) that neutrophilic and eosinophilic myelocytes are 
of two types — a large type (macromyelocyte), and a small type 
(micromyelocyte) ; (/) that the granules of the eosinophilic myelo- 
cytes are of a bluish-purple color, whereas the eosinophilic poly- 
morphs have bright red granules; (g) that many of the mononuclear 
neutrophilic cells have a bean-shaped nucleus — the metamyelocytes. 
Further note the occurrence (h) of large mononuclear leukocytes, 
devoid of neutrophilic, eosinophilic and mast-cell granules, but 
presenting a well-staining basophilic spongioplasm — leukoblasts; 
(i) of cells showing the general structure of a small mononuclear 
leukocyte, but of larger size and often showing the cytoplasm 
gathered in knobs on the periphery — macrolymphocytes; stained 
with an azure mixture these could be shown to be lymphoidocytes; 
( j) true small monos and large monos are very scarce ; (k) polymor- 
phonuclear basophiles — mast cells — and corresponding mononuclear 
forms are very common — some with fine granules, others with coarse 
granules. Nucleated red cells, mostly normoblasts, some undergoing 
mitosis, will also be met with. 

Illustrate the Above Points. — Attempt a differential count of 200 cells. 

Answer the Following Questions in Writing. — What is a lymphoido- 
cyte, a leukoblast and a myelocyte, (a) structurally considered, (6) 
ontogenetically considered? Draw a schema, showing the inter- 
relationship between the different kinds of leukocytes themselves 
and the red cells. Where do the lymphocytes originate? where the 
granulocytes? In what manner does the blood picture of the ordi- 
nary lymphocytosis differ from that seen in acute lymphatic leuke- 
mia, so-called? In what manner does hyperleukocytosis of the 
septic type differ from that seen in chronic myelogenous leukemia? 
show the points of difference in a tabular form. Does the appear- 
ance of myelocytes per se warrant the diagnosis of myelogenous 
leukemia; if not, why not? What is meant by myelocytosis or 
myelemia ? 

Reading. — 

Lesson 9. — Students to be supplied with blood smears from (a) 
a case of chlorosis; (6) a case of secondary anemia, due to a severe 



28 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



streptococcus infection; (c) a case of secondary anemia due to cancer 
of the stomach; (d) a case of pernicious anemia (Biermer type), all 
stained with Jenner's stain. Study the smears with the oil-immersion 
lens, in reference to the following points, arranging your findings in 
tabular form : (a) The amount of coloring matter in the individual 
cell — whether there is normocytochromia, hypocytochromia, or 
hypercytochromia; to this end pay attention only to cells that have 
not been flattened out, but show the central pale area; (b) abnormal 
variation in size — auisocytosis and its extent; (c) abnormal varia- 
tion in form — poikilocytosis and its extent; (d) tendency to oversize 
(macrocytosis) ; (e) tendency to oval form ; (/) the occurrence of 
polychromatophilic red cells — their relative number; (g) the occur- 
rence of stipple cells — cells undergoing so-called granular degenera- 
tion — their relative number; (h) the occurrence of normoblasts; (i) 
of megaloblasts; (j) the number of blood platelets, whether in- 
creased or diminished. Draw all These Features. 

Now examine the smears with the low power and note whether 
the leukocytes are present in normal numbers or whether they are 
increased or diminished. In fine do a differential leukocyte count. 

Answer the Following Questions in Writing. — What is the color 
index (a) in chlorosis; (b) in secondary anemia; (c) in pernicious 
anemia? What is meant by (a) hypocytochromia; (6) hypercyto- 
chromia? What is meant by (a) anisocytosis, (6) poikilocytosis; 
(c) macrocytosis; (d) polychromatophilia or polychromasia ; (e) an 
erythroblast? What does polychromatophilia indicate? is it a regen- 
erative or a degenerative symptom? (/) What is a stipple cell? 
is it a regenerative or a degenerative symptom? Under what path- 
logical conditions are stipple cells notably met with? What are the 
cardinal features (a) of a normoblast? (6) of a megaloblast? What 
conclusion may be drawn from the presence of erythroblasts in the 
circulating blood ? What is the prognostic significance of the megal- 
oblast as compared with the normoblast? What relation does the 
megaloblast bear to the normoblast? from what cell is it derived? 
What is meant by a blood platelet? what is the origin of the plate- 
lets? under what conditions are they notably increased? in what type 
of anemia are they notably diminished in number? In distinguish- 
ing between Biermer's anemia and the pernicious type of anemia 
seen in cancer cases, other things being equal, in what direction 
would leukopenia point? which cells would be deficient and which 
relatively increased? 

Reading. — 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 29 



Lesson 10. — Apparatus and Reagents Required. — Hagedorn needles; 
sterilized gauze sponges in pairs, covered with muslin; small test- 
tube racks for agglutination tubes — one for every man; a dozen 
agglutination tubes per student, made from glass-tubing one- 
fourth inch inside diameter, each two inches long (these can be 
prepared by the student himself); clean slides and cover-glasses; 
vaselin in tubes — one for every two men; alcohol in individual 
bottles; capillary pipettes — these also can be prepared by the 
students themselves; rubber nipples to fit the capillary pipettes; 
Dunham solution cultures — not more than twenty-four hours old, 
grown at room temperature, of the typhoid bacillus, of the paraty- 
phoid A and paratyphoid B — a culture of each for every six men ; cor- 
responding antisera, diluted so as to give a definite agglutination reac- 
tion in one hour, at room temperature, when the microscopic method 
is applied — 10 c.c. for every six men; a high-titer serum against each 
one of the organisms mentioned — in such dilution as to give a 
macroscopic reaction in five minutes — 3 c.c. for every six men. A 
bichloride or lysol basin to be provided for every twelve men (a) for 
purposes of hand disinfection, (b) for disinfecting apparatus used 
at the end of the lesson; glass pencils — one for each student. Have 
Bunsen burners available. 

Directions to the Student. — Charge agglutination tubes with about 
1 c.c. of the three cultures given out and label correspondingly; 
charge another set of tubes each with 1 c.c. of the corresponding 
antisera — label, noting the degree of dilution; charge a further 
set of tubes with about 0.5 c.c. — 8 drops — of the three high-titer 
sera — label, noting degree of dilution. Have a separate pipette 
for each tube, marked with glass pencil. 

Experiment A. — Macroscopic Slide Agglutination Method. — On 
a slide mount, in a row, a large drop from each one of the cultures 
marked, T, PTa and PTb; place two similar rows of drops of the 
same organisms below these. To each drop of the top row now add 
one drop of the high-titer antityphoid serum; to each one of the 
second row add one drop of the high-titer antiparatyphoid-A 
serum and to each drop of the third row one drop of the high-titer 
antiparatyphoid-B serum. Place the slides on the stage of the 
microscope and view them by looking at the mixtures, with the 
naked eye, on a slant, and then with the low power of the micro- 
scope. Clumps will begin to form in some of the drop mixtures within 
a few minutes; note that this will occur first and become most 
marked in the homologous mixtures, viz., in the typhoid-anti- 
3 



30 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



typhoid drop, in the p-typhoid-a-anti-p-typhoid-a and in the para- 
typhoid-b-anti-p-typhoid-b drops, while in the crossed mixtures 
clumping will either not occur at all or develop much later and then 
be less marked. In this manner it is evident that the nature of an 
unknown antiserum or an unknown bacillus may be identified if the 
corresponding reagent is available. 

Draw these appearances and append explanatory legends. 

Experiment B. — Microscopic Slide Agglutination Method. — 
Prepare two vaselin rings on each one of three slides; the walls 
should be of uniform height — one-eighth inch; leave no gaps; label 
slide (1) T; slide (2) PTa; slide (3) PTb. Charge each vaselin 
chamber of slide 1 with a drop of the typhoid culture; add to one a 
drop of the antityphoid serum, of moderate titer, and to the other a 
drop of saline. Adjust a cover-glass, so as to be everywhere in con- 
tact with the vaselin wall; press the cover down evenly and gently 
until it touches the drop mixture below. Prepare the other two 
slides in a similar manner with the corresponding organisms and anti- 
sera. Examine the mixture with the low power of the microscope 
with the condenser out, using the flat mirror and a subdued light; 
the saline-bacillary drops serve as a check on the corresponding 
bacillary antisera mixtures, to indicate whether the bacillary 
emulsions were homogeneous to begin with, and to call attention 
to the occurrence of auto-agglutination, which, of course, would 
invalidate the significance of an agglutination reaction in the 
corresponding bacillary antiserum specimen. With the low power 
of the microscope the focussed drop mixtures will appear evenly 
granular. Set the specimens aside and reexamine at intervals of 
ten minutes ; make your final readings at the end of one hour. You 
will then note that the background of the bacillary-antisera, in 
contradistinction to the bacillary-saline specimens, will no longer 
appear uniformly granular, but that "wall patterns" composed of 
masses of agglutinated organisms with clear interspaces will have 
formed. Make drawings to illustrate appearances at the beginning 
and the ending of the hour, (a) as seen with the low power; (6) as 
seen with the middle power. The adjustment of the latter must be 
carried out with care, so as not to force the mixture through the 
vaselin wall. Note with the middle power that the previously 
actively motile organisms have become immobilized by the anti- 
sera, while in the saline controls the motility remains as in the 
beginning. Draw and append explanatory legends. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 31 



Experiment C. — Macroscopic Test-tube Method. — Charge three 
agglutination tubes with 0.5 c.c. each of the three bacillary emulsions 
and label accordingly (1) T; (2) PTa; (3) 1Tb; add to "each bacil- 
lary emulsion 0.5 c.c. of the corresponding antiserum of moderate 
titer; put up corresponding saline control tubes and crossed mix- 
tures — e. g., 0.5 c.c. of typhoid emulsion plus 0.5 c.c. of anti-para- 
typhoid serum; close all tubes with little cotton stoppers and set 
them aside at room temperature until the following day. Then 
note that a granular sediment covered by a clear supernatant fluid 
will have formed in the homologous mixtures, while the saline controls 
remain turbid and the cross mixtures show only an inconclusive 
reaction. Draw the appearances and append explanatory legends. 

Answer the Following Questions in Writing. — What is meant by 
the terms (a) antigen, (6) antibody? Give classification of the 
different antibodies and the corresponding antigens, arranged in 
tabulary form. How would you define an agglutinin? What 
term is applied to the corresponding antigen? Who discovered 
the agglutinins? In what manner did Widal's name become con- 
nected with the history of the agglutinins? Is the agglutination 
reaction used for diagnostic purposes outside of the study of typhoid 
fever? Why is it necessary to dilute the patient's serum in doing 
the Widal test? How early in the disease does it appear? How 
long does it persist after recovery? Does a positive Widal invari- 
ably indicate that the patient is suffering at the time or has been 
suffering in the past from typhoid fever? Is it essential that the 
bacilli used in the test be alive? How may they be preserved for 
agglutination purposes? Will the serum of a paratyphoid patient 
agglutinate typhoid bacilli? How would you distinguish between 
a typhoid and a paratyphoid case serologically? What is meant 
by spontaneous agglutination? How would you proceed to rule 
out spontaneous agglutination in doing the agglutinin test in a 
suspected case of paratyphoid fever? 

Reading. — 

Lesson 11. — Apparatus and Reagents Required. — Same as for 
Lesson 10. In addition supply two specimens of fecal material- 
particles the size of a pea, emulsified in about 10 c.c. of saline, one 
of which is to contain typhoid bacilli in addition to the usual flora; 
the other should contain no typhoid organisms. Do not indicate on 
the label which is which, but mark x and y. Supply every two 
students with one Drigalski-Conradi plate, one Endo-plate. a Bril- 



32 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



liant green plate and a Russell plate; use large plates and fill them 
to a depth of at least 3 mm. Have also available a colon culture, 
besides the typhoid culture. 

Experiment A. — Collect about 10 drops of blood from your 
working partner's ear, using a small agglutination tube for the 
purpose, dipping up the drops as they are squeezed out. Allow the 
blood to clot; loosen the clot from the walls of the tube with your 
nichrome wire or a sealed capillary pipette; centrifugalize; pipette 
off the supernatant serum and dilute with saline 1 to 25, using 
an agglutination tube; label 1 to 25. With this diluted serum do 
the microscopic slide agglutination test and the macroscopic test- 
tube test, as described in the previous lesson. Read the result of the 
first at the end of one hour and that of the second the next day. 

Answer the Following Questions in Writing. — What result did you 
obtain in examining your partner's blood? If positive did you 
ascertain whether he has had typhoid fever in the past and when? 
If he gives no history of typhoid, did you ascertain whether he has 
been vaccinated against typhoid and could that give rise to a positive 
reaction? Does a Widal reaction occur in typhoid carriers? If you 
found a positive Widal in a female, a number of years after an attack 
of typhoid, and if you also obtained a history of gall-stone attacks 
in the individual, what would your course of procedure be and 
why? 

Experiment B. — With a sterile sealed capillary pipette divide 
each one of your culture plates into four quadrants and mark these 
with a blue pencil on the bottom of the plate. Place a minute drop 
of the typhoid culture (in bouillon) on quadrant I and spread it out 
over the entire quadrant with your nichrome wire or a bent and 
sealed capillary pipette; smear quadrant II with a droplet of the colon 
culture, quadrant III with the fecal emulsion x and quadrant IV with 
the fecal emulsion y. Cover the plates, turn them over and incubate 
until the next day. 

Answer the Following Questions in Writing. — What is the com- 
position of (a) the Conrad i-Drigalski medium; (b) of the Endo 
medium; (c) the brilliant green medium; (d) the Russel medium? 
What role does the anilin dye play in a, b and c respectively ? What 
is Andrade's indicator? what does it indicate? 

Reading. — 

Lesson 12. — Continuation of Lesson 11. — Reagents Required. — The 
same as in the preceding lesson, plus four tubes of glucose bouillon 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 33 



and of litmus milk for each man. Examine the plates which 
you incubated yesterday. Note and draw with colored pencils 
the appearances of the typhoid and colon colonies in quadrants 
I and II. Carefully examine the colonies in the remaining quad- 
rants and by comparison with those of I and II ascertain which 
fecal specimen contained typhoid bacilli and which was free of them. 
Draw these appearances also. Prove that the colonies which you 
suspect of being typhoid are in reality so (a) by the macroscopic 
slide agglutination method, (b) by culture in glucose bouillon and in 
litmus milk. Do controls with the colonies obtained from quadrants 
I and II, as well as with those from the fecal non-typhoid quadrant 
and the non-typhoid colonies of the fecal typhoid quadrant. 

In doing the macroscopic slide agglutination test mount a series 
of good-sized drops of the high titer antityphoid serum (1 to 100) in 
one row (top) and a second row of saline drops below this, so that a 
saline drop corresponds to a serum drop. Fish a colony that you 
wish to examine with the nichrome loop, emulsify it in the saline 
drop and transfer a bit of the emulsion to the serum drop; or 
emulsify a colony of the same kind in the latter; label your speci- 
mens with the glass pencil. 



o 


o 


o 


o 


FT 


FNT 


T 


C 


o 


0 


o 


o 



serum row 
saline row 



FT = fecal typhoid; FNT = fecal non-typhoid; T = typhoid; C = colon. 

The drops should not be too small, otherwise they will dry 
up before a result can be obtained. Observe the drops in the top 
row with the naked eye on a black background — stage of the micro- 
scope — or with the low power and note the development of clumps 
of agglutinated bacilli. Illustrate the final appearance of all the 
drops and append appropriate legends. 

Answer the Following Questions in Writing. — What is meant by a 
typhoid carrier? Do typhoid bacilli occur in the fecal content- of 
typhoid patients as a rule? If so, how long into convalescence do 
they persist? Do they usually persist? What is meant by a chronic 
carrier? What is meant by an active, and what by a passive carrier ''. 
What relationship exists between gall-bladder infection and the 
carrier state? Are female carriers more common than male carriers, 



34 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



or vice versa? Which are more common, fecal or urinary carriers? 
Is the elimination of organisms in fecal carriers continuous or 
intermittent? 

What is the reason of the difference in the appearance in your 
plates of the typhoid as compared with the colon colonies: (a) on 
the Drigalski plate, (6) on the Endo plate, (c) on the brilliant green 
plate, (d) on the Russell plate? 

W T hat is the difference in behavior of the typhoid as compared 
with the colon bacillus (a) in glucose bouillon, (6) in litmus milk? 
What other bacilli occurring in the feces are alkali producers? How 
would you distinguish between them and the typhoid bacillus? 
What other bacilli besides the colon bacillus, occurring in the feces 
are acid producers? 

Reading. — 

Lesson 13. — Apparatus and Reagents Required. — Furnish every 
student with one agglutination test-tube rack holding a dozen tubes; 
four test-tubes of ordinary size; one clean tumbler; one 1 c.c. 
pipette, graduated in tots; a dozen capillary pipettes with rubber 
nipples; a wax pencil. 

Supply every six men with 250 c.c. of sterile 0.9 per cent, saline; 
50 c.c. of a 2.5 per cent, washed sheep corpuscle emulsion; 10 c.c. of 
diluted fresh complement (1 to 10), or using acetated complement 
(one part of guinea-pig serum, treated with one and a half part of 
a 10 per cent, solution of c. p. sodium acetate in 0.9 per cent, saline), 
dilute 2.5 c.c. of the acetated complement with 7.5 c.c. of saline; 
10 c.c. diluted amboceptor — strength to equal three times that of 
the titer; 10 c.c. of diluted cholesterinized antigen — strength to 
equal two-thirds that of the titer; 2.5 c.c. of an actively fixing 
syphilitic serum and a like quantity of a non-fixing serum; 10 c.c. 
and 1 c.c. pipettes graduated in to" and tito" c.c. respectively to 
accompany the reagents. 

Have available also one water-bath, at 56° C, and one water-bath 
at 37° to 40° C, provided with racks for tubes; an electric or high- 
speed water-power centrifuge, preferably with many arms (labora- 
tory technician should supervise work at centrifuge) . 

Directions to the Student. — Provide yourself from the stock 
reagents set out for every six men with 40 c.c. of saline — put this 
in your tumbler; 8 c.c. of sheep corpuscle emulsion, put this in a 
test-tube; 1.5 c.c. each of diluted complement, amboceptor and 
antigen; and 0.4 c.c. each of the syphilitic (I) and the non-syphilitic 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 35 



(II) serum; put these into your small test-tubes; now proceed with 
the following experiment: 

Experiment A. — Mark four agglutination tubes serially 1, 2, 3 
and 4. Charge the tubes as follows: (1) with 0.2 c.c. corpuscle 
emulsion and 0.4 c.c. saline; (2) with 0.2 c.c. corpuscle emulsion, 
0.2 c.c. diluted complement and 0.2 c.c. saline; (3) with 0.2 c.c. 
corpuscle emulsion, 0.2 c.c. diluted amboceptor and 0.2 c.c. saline; 
(4) with 0.2 c.c. corpuscle emulsion, 0.2 c.c. diluted amboceptor 
and 0.2 c.c. diluted complement. 1 Incubate in the water-bath at 
37° to 40° C, or in the absence of such, in your upper vest pocket. 
Shake every few minutes. Note the result at the end of fifteen 
minutes. The occurrence of hemolysis is indicated by laking of the 
blood cells. Centrifugalize your tubes and note the appearance 
of the fluid, whether colorless or blood-tinged, and the appearance 
or non-appearance of a sediment of non-hemolized red cells, at the 
bottom. Arrange your findings in tabulary form, stating under 
the heading "results" whether hemolysis has or has not occurred; 
whether it is complete or not, e. g. : 

Components. Period of incubation. Result. 

Tube (1) corpuscles -f- saline. Fifteen minutes. No hemolysis. 

Experiment B. — Repeat experiment A-4, (a) using complement 
that has been previously kept at a temperature of 56° C. for fifteen 
minutes and (b) using amboceptor that has been treated in the 
same manner. Note the results and compare with those obtained 
sub A-4. 

Experiment C. — First Step. — Place your 0.4 c.c. portions of 
syphilitic and non-syphilitic serum in agglutination tubes, marked 
I and II respectively, and keep in the 56° C. water-bath for fifteen 
minutes. 

Second Step— Add 2 c.c. of the sheep corpuscle emulsion to each 
tube; shake well and incubate at 37° to 40° C. for ten minutes. 

Third Step. — Centrifugalize until the supernatant fluid is per- 
fectly clear; pipette this off by the aid of a clean capillary pipette, 
marking the tubes I and II as before. 

Fourth Step. — Mark four agglutination tubes IA, IC, IIA and 
IIC respectively. Charge IA with 0.2 c.c. of serum I, treated as 
just directed; add 0.2 c.c. of the diluted antigen and 0.2 c.c. of 

1 If suitable graduated pipettes are not available, use capillary pipettes, allowing 
3 drops for 0.2 c.c; wash out the pipette with a few changes of saline before taking 
up another reagent. 



36 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



the diluted complement. Charge IIA in a corresponding manner, 
using serum II in the place of serum I. Tube IC is the control to 
IA and receives 0.2 c.c. of the serum, 0.2 c.c. of the diluted com- 
plement and 0.2 c.c. of saline instead of the antigen. Tube IIC is 
correspondingly charged, using serum II instead of serum I. The 
four tubes are now incubated for fifteen minutes at body tem- 
perature. 

If doubly and trebly tiered racks are available the A tubes are 
arranged on one tier and the C tubes immediately behind the others ; 
they may then be charged in situ, as shown in the diagram : 



o o o o o o A row with antigen. 

o o o o o o B row in which a second antigen may be used, 
o o o o o o C row without antigen. 



At the expiration of the fifteen minutes each tube — both of the 
A row and the C row — now receive 0.2 c.c. of the diluted amboceptor 
and 0.2 c.c. of the corpuscle emulsion. The tubes are well shaken 
and returned to the water-bath, where they remain for fifteen 
minutes. At the end of that time it will be noted that the contents 
of the C tubes are entirely hemolyzed and perfectly clear. Centrif- 
ugalize the A tubes and compare the result in tube I with that 
obtained in tube II. Note the color of the supernatant fluid and the 
presence or absence of non-hemolyzed red cells at the bottom. 
Arrange your findings in tabulary form; under the heading 
"Results," state whether or not hemolysis has occurred, whether 
there is none whatever, or whether it is partial or complete, e. g.: 



Serum 
1 to 6. 


Comple- 
ment 
1 to 10. 


Antigen 
1 to 10. 


37° C. 
utes. 


Ambo- 
ceptor 
1 to 1000. 


Corpuscle emulsion 
2 . 5 per cent. 


37° C. 

tutes. 


Result 








„, B 






5 




0.2 c.c. A 
0.2 c.c. C 


0.2 c.c. 
0.2 c.c. 


0.2 c.c. 
0.2 c.C. 


Incubate 
for 15 


0.2 c.c. 
0.2 c.C. 


0.2 c.c. 
0.2 C.C. 


Incubat* 
for 15 


No hemolysis. 
Complete " 



Answer the Following Questions in Writing. — What is the source 
of the complement usually employed in the complement-fixation 
tests? How is the reagent prepared, ready for use? What is ace- 
tated complement and how is it prepared? How is the sheep cor- 
puscle emulsion prepared? What is the source of the antisheep 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



37 



amboceptor? How is it obtained and how is it kept? What is 
meant by the titer of (a) the amboceptor, (6) the complement? 
What is the effect of heating at 56° C. upon complement? How is 
the amboceptor affected under like conditions? Illustrate dia- 
grammatically the manner in which hemolysis is produced and the 
role played by complement and amboceptor. In doing the Wasser- 
mann test, why do you first heat the serum at 56° C? Why do you 
subsequently treat it with a sheep-cell emulsion? What is meant 
by an antisheep hemolytic system? On what principle is the 
Wassermann test based? What role is played in the test, respec- 
tively, by the patient's serum? the antigen? the complement, the 
amboceptor and the corpuscles? What is the object of the incu- 
bation at 37° C. after bringing the patient's serum, the antigen and 
the complement together? Why do you finally add the sheep 
corpuscles and the amboceptor? Why does hemolysis not occur 
in the end if the patient's serum was syphilitic? and why does it 
take place if the serum was non-syphilitic? What does the term 
"complement fixation" denote in connection with the test in 
question? What is the active principle of the antigen used in the 
Wassermann test? What is the source of the antigen in question? 
How is it prepared? What is meant by cholesterinized antigen? 
What is the idea underlying the preparation of Xoguchi's antigen? 
What is meant by the titer of the antigen? 
Reading. — 

Lesson 14. — Apparatus and Reagents Required. — For every six 
men furnish of alcoholic beef-heart extract 50 c.c; of cholesterinized 
beef-heart extract the same quantity; 250 c.c. of saline; 50 c.c. of a 
2.5 per cent, emulsion of washed sheep corpuscles; 30 c.c. of diluted, 
fresh complement (1 to 10), or using acetated complement dilute 
2.5 c.c. with 7.5 c.c. of saline; 30 c.c. of antisheep amboceptor, 1 
to 1000. 

Every student to be supplied with two dozen agglutination 
tubes; an agglutination test-tube rack; capillary pipettes; rubber 
nipples; 1 c.c. pipette graduated in hundredths; a wax pencil, as 
in Lesson 13. 

A water-bath kept at 56° C, one at 37° to 40° C, and an 
electric centrifuge should also be available. As in the preceding 
lesson the technician should supervise the work at the centrifuge. 

Directions to the Student. — Provide yourself from the stock re- 
agents set out for every six men, with -10 c.c. of saline; S c.c. of 
4 



38 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



sheep corpuscle emulsion; 5 c.c. of diluted complement; 5 c.c. of 
antisheep amboceptor and 6 c.c. each of the plain beef-heart extract 
and the cholesterinized extract; mark all 'your reagents with the 
wax pencil. 

Experiment A. — Determine the Titer of the Amboceptor. — Of the 
1 to 1000 dilution furnished, prepare subdilutions as follows: Mark 
six agglutination tubes 1000, 2000, 3000, 4000, 5000 and 6000 
respectively; place them in your rack (first row) and charge each 
one with 0.2 c.c. of the 1 to 1000; to the second tube add 0.2 c.c. 
saline; to the third 0.4 c.c, to the fourth 0.6 c.c, to the fifth 0.8 
c.c and to the sixth 1.0 c.c. Set up a second row of five tubes, marked 
from 2000 to 6000 and place these in the second row behind those of 
the first; move your 1 to 1000 tube, as it is, to the second row to 
its proper place. Then transfer 0.2 c.c of each dilution in the front 
row, to the corresponding tube in the back row (see diagram). 

1 to 1000 1 to 2000 1 to 3000 1 to 4000 1 to 5000 1 to 6000 

f First row for holding 

O O O O O O j the original dilu- 

[ tions. 

f Second row for hold- 

O O O O O O J ing 0.2 c.c. each of 

I the corresponding 
[ dilutions. 

To each tube of the second row add 0.2 c.c. of the diluted comple- 
ment and 0.2 c.c of the corpuscle emulsion; shake well and incubate 
for fifteen minutes in the water-bath at 37° C, shaking from time 
to time. At the expiration of the fifteen minutes examine your 
tubes and note the extent to which hemolysis has taken place. The 
highest dilution which has cleared absolutely indicates the titer 
of the amboceptor. In the actual Wassermann test or in titrating 
the antigen (see below) three times the titer strength is used, e. g., 
if the titer was 1 to 5000 we should use a dilution that is three times 
as strong; in the present instance this would be £_o_^_o = — \ 
to 1666. Every remaining cubic centimeter of the 1 to 1000 dilution 
would hence be further diluted by adding 0.66 c.c of saline. Do 
this and label the resultant amboceptor dilution correspondingly. 

Experiment B. — Determine the Titer of Both Antigens as follows: 
Set up a series of six agglutination tubes and mark them 3 to 10, 
2.5 to 10, 2 to 10, 1.5 to 10, 1 to 10, 0.5 to 10. Charge the tubes 
respectively with 3.5, 3.75, 4.0, 4.25, 4.5, 4.75 c.c. of saline, and add 
of the antigen to be examined 1.5 c.c to the first, 1.25 c.c. to the sec- 
ond, 1.0 c.c to the third, 0.75 c.c to the fourth, 0.5 c.c to the fifth 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 39 

and 0.25 c.c. to the sixth, using the 1 c.c. pipette, graduated in 
hundredths, washing it out well with saline after use. Mix the con- 
stituents well. Mark a second set of tubes in the same manner as 
the first set and arrange them in the rack in the second row, so as to 
correspond to those in the first. Transfer from the mixtures in the 
first row 0.2 c.c. to the corresponding tubes in the second row, 
starting with the highest dilution, i. e., the 0.25 to 4.75 which 
represents a dilution of 0.5 to 10. Add to each tube of the second 
row 0.2 c.c. of the diluted complement and 0.2 c.c. of saline; mix 
well and incubate for ten minutes at 37° C; then add 0.2 c.c. of 
amboceptor, using three times the titer strength and 0.2 c.c. of the 
sheep corpuscle emulsion; mix well and reincubate for fifteen min- 
utes; then centrifugalize and note the results, viz., whether com- 
plete hemolysis has taken place in any one of the tubes. The 
highest concentration of the antigen dilution in which complete 
hemolysis was obtained represents the titer. In the actual Wasser- 
mann test the antigen is used in a concentration corresponding to 
two-thirds the titer dose. Write down your results in tabulary 
form; indicate the titer and the concentration to be used in the 
actual Wassermann Test. 

After having determined the titer of the plain beef-heart extract, 
ascertain that of the cholesterinized antigen, proceeding in the same 
manner. Wash out your tubes, first with water, then with saline. 

Answer the Following Questions in Writing. — The titer of an 
antigen was 1.5 in 10; how much saline and undiluted antigen 
respectively would you use to make up a total bulk of 5 c.c. for 
actual work? You are to examine 10 sera for the Wassermann 
reaction and to use 0.4 c.c. of every diluted reagent, as standard 
volume, employing two antigens; the titer of your amboceptor is 1 
to 3000; how many cubic centimeters of how strong a dilution 
are you going to make up to cover your needs? Give your 
calculation in detail. How many cubic centimeters of uative 
complement will you need, and how many cubic centimeters 
of corpuscle emulsion? How is acetated complement prepared? 
Starting with acetated complement, how many cubic centi- 
meters would you need and how much saline would you add to 
examine the ten sera in question? How much hashed beef -heart 
would you use to make up 200 c.c. of plain heart extract? 
How much 1 per cent, cholesterin solution, and how much of the 
plain alcoholic extract would you use to make up 100 c.c. of the 
mixture? How would you ascertain whether a rabbit that you have 



40 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

been immunizing against sheep cells is ready to be killed? How 
would you store its serum? Of what strength should your stock 
solution be? How would you keep the latter? Why does Noguchi 
recommend the use of an antihuman hemolytic system? How do 
others overcome the same objection attaching to the use of the anti- 
sheep system? What does the writer mean by an instantaneous 
Wassermann reaction? What would you do if a patient's serum 
only showed partial complement fixation after fifteen minutes' 
incubation? How would you express your findings if complement- 
fixation resulted only after forty-five minutes' incubation? How 
would you express your findings if you obtained an instantaneous 
reaction ? 
Reading. 

Lesson 15. — Transfusion Test. — Apparatus and Reagents Re- 
quired. — Sterilized gauze sponges ia pairs, wrapped in muslin; 
Hagedorn needles; individual bottles of alcohol (2 oz.); agglutin- 
ation tubes, such as were used for collecting blood for the Widal 
reaction — two for each student; 0.9 per cent, saline, containing 
1.5 per cent, sodium citrate, 10 c.c. for every six men; 0.9 per cent, 
saline, 150 c.c. for every six men; 2 c.c. of an antihuman serum 
(1 to 1000) ; two capillary pipettes with rubber nipple for every man; 
clean slides and cover-glasses; vaselin in tubes, one for every two 
men; wax pencils, nichrome wire; an incubator at 37° to 40° C; 
electric centrifuge. 

Directions to the Student. — Supply yourself from the stock reagents 
with about 1 c.c. of the citrate solution; place this in one of your 
agglutination tubes; take about 25 c.c. of saline and keep this in a 
little whiskey glass or beaker. Mark your reagents with the wax 
pencil. Puncture your working partner's ear with the Hagedorn 
needle, after cleaning it with alcohol and drying it, in the usual 
manner. Collect about eight or ten drops of blood in a dry agglutin- 
ation tube; after clotting has occurred, loosen the clot with your 
nichrome wire and centrifugalize. Also collect about five or six 
drops of the same person's blood in the little tube, containing the 
citrate solution. Regard yourself as the recipient of your working 
partner's blood and his serum and corpuscles accordingly as the 
donor's. Your working partner will similarily regard himself as 
the recipient of your serum and corpuscles, and you accordingly 
as the donor. Mark serum and corresponding corpuscles with 
the name of the individual from whom they were obtained. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 41 

Prepare a couple of vaselin rings on the slide, side by side, and 
mark them R-s (recipient's serum) and D-c (donor's corpuscles) 
and D-s (donor's serum) and R-c (recipient's corpuscles). By 
the aid of one of your capillary pipettes charge the little vaselin 
chambers as indicated, mixing two large drops of the respective 
serum that is called for, with one small drop of the corresponding 
corpuscle emulsion. Wash out the pipette after the use of every 
reagent, with a few changes of saline from your little beaker. Adjust 
a cover-glass over each ring, as in your Widal work, pressing it into 
position, until it touches the drop and is everywhere in contact with 
the vaselin ring. Examine the specimen with the middle power of 
your microscope, and note that the red cells all show their greenish- 
yellow color and that there are no blood shadows (colorless rings) or 
at most a few isolated ones. Incubate the slide for thirty minutes 
and reexamine. If either serum contained hemolysins the cor- 
puscles of the corresponding mixture will either have disappeared 
entirely or blood shadows will be present in large numbers — exten- 
sive hemolysis may be recognized with the naked eye by the occur- 
rence of extensive clearing of the specimen. If either serum con- 
tained agglutinins the corpuscles of the specimen will be gathered 
in clumps which are usually visible with the naked eye. If neither 
hemolysins nor agglutinins are demonstrable the blood of the donor 
may be regarded as entirely suitable for transfusion purposes, pro- 
viding of course the donor is not syphilitic nor suffering from 
bacteriemia. Slight agglutination may be disregarded. If extensive 
agglutination occurs it is advisable to seek another donor. If 
hemolysis occurs the donor should of course be rejected. 



m 

i i 


i 

p 

i i 


T 


T 



R-s + D-c D-s + R-c 

If in the above experiment the sera examined were free from any 
hemolytic action prepare a vaselin ring on another slide and mount 
a mixture of one drop of your corpuscle emulsion with one drop of 
the anti-human (rabbit) serum supplied to your group; adjust a 
cover-glass and incubate for a few minutes; examine with the low 
power at frequent intervals, and as soon as you see any evidence 
of hemolysis look for blood shadows with your middle power. 



42 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



Answer the Following Questions in Writing. — What technical term 
is applied to hemolysins which will hemolyze the red cells of an 
animal of the same species? Why would the presence of such hemoly- 
sins in the serum of either the recipient or the donor, in connection 
with a contemplated transfusion, be objectionable? Why would 
the presence of agglutinins under like conditions be objectionable? 
What is meant by hemoglobinemia? What is meant by auto- 
hemolysins? What relation exists between the occurrence of 
paroxysmal hemoglobinuria and the activity of autohemolysins? 

Reading. — 

Lesson 16. — Blood-sugar Estimation. — Apparatus and Reagents 
Required. — Provide every two students (working partners) with the 
following: 25 to 30 c.c. of a saturated aqueous solution of picric 
acid; a similar quantity of a 10 per cent, aqueous solution of sodium 
carbonate; a tumblerful of water; 1 or 2 c.c. of a 2.0 per cent, 
solution of sodium fluoride; a 1 c.c. pipette, graduated in tenths; a 
capillary pipette with rubber nipple; a 10 c.c. pipette graduated 
in tenths; a boiling test-tube of similar capacity — of as wide a 
diameter as possible; a Sahli hemoglobin ometer calibrated tube; 
a standard color tube both of the A and the B type, such as are fur- 
nished with the Kuttner microcolorimeters; one colorimeter stand of 
the latter type. Bunsen burners and centrifuge. 

Supply every six students with a few cubic centimeters of 
ordinary oxalated human blood, which may be collected in vacuum 
tubes containing a little powdered sodium oxalate, or by the open 
method, allowing blood to run through a needle from a vein directly 
into a tube containing a little oxalate or citrate; in either case the 
mixture should be well shaken, immediately after the blood has been 
drawn; mark this (I). If possible secure a specimen of diabetic 
blood also (II). 

Directions to the Student. — Of specimen I place 0.2 c.c. in the 
10 c.c. graduated test-tube, rinse the pipette two or three times 
with water, adding the rinsings to the blood; dilute with water 
to the 1 c.c. mark and add picric acid solution up to the 2.5 
c.c. mark. Shake well and centrifugalize. Pipette off the clear 
supernatant fluid and place 1 c.c. in the boiling test-tube furnished 
you. Be careful, picric acid is poisonous. Boil down to two or 
three drops, shaking all the time, so that the tube does not crack. 
Add 0.5 c.c. of the 10 per cent, solution of sodium carbonate, having 
washed out your 1 c.c. pipette, after using it to measure off the 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 43 



picric acid mixture. Boil again until the last few drops undergo 
crystallization on removal from the flame; note the change in color 
to brownish red. Dissolve the residue with a few drops of water, 
by the aid of a little heat, and transfer the solution to the calibrated 
tube of the colorimeter; rinse with a few drops of water at a time 
and add the rinsings to the solution ; dilute to the 50 mark and com- 
pare the color with the standards A and B. If it is darker than A, 
but lighter than B, the former is used as standard; the mixture is 
then diluted with water, as in your hemoglobin estimation, until the 
colors match. To ascertain this point it is well to compare them, 
while the tubes stand in the little colorimeter, the slide of the latter 
being lowered down to the bleeding prism. Read off the figure 
reached; divide by 1000; the resultant number indicates the per- 
centage of sugar in the specimen examined. If the darker tube B 
is used the result must be multiplied by 2. 

Repeat the examination with specimen II, which was obtained 
from a diabetic patient. In this case start your work, using 0.1 
instead of 0.2 c.c. of blood and multiply all your figures in the end 
by 2. After completing your work, carefully wash out all your 
tubes and pipettes — using plain water — and if necessary a little 
alcohol. In noting your results indicate clearly how much blood 
you used to start with, what your readings were and how you 
arrived at your final figures. 

Repeat the examination with your working partner's blood, 
collecting the necessary amount from a free ear-puncture and 
allowing the blood to flow into a little tube containing a drop of 
a 2 per cent, solution of sodium fluoride. 

Answer the Following Questions in Writing: What is the normal 
sugar content of the blood? \Yhat is meant by hyperglycemia? 
Under what conditions does this occur? Contrast the findings 
obtained in a case of alimentary hyperglycemia following the 
administration of 100 grams of dextrose, with the result that you 
would be most liable to obtain in a diabetic under like conditions. 

Reading. — 

Lesson 17. — Blood Urea Estimation. — Apparatus and Reagents 
Required. — On the day of the experiment furnish every two students 
with two cylinders of approximately 150 c.c. capacity each; two 
Erlenmeyer flasks of 200 c.c. capacity; two Folin absorption tubes; 
two calcium chloride tubes, filled with cotton; and the necessary 
glass tubing, rubber tubing and rubber stoppers to set up the 



44 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



arrangement shown in the accompanying illustration. Furnish 
also a Mohr's burette with stand. 



c <y 



Pump e- 




Supply every six men with one absorption bottle (see D in illus- 
tration), containing 30 per cent, sulphuric acid; so also 500 c.c. ^ 
sulphuric acid, 500 c.c. sodium hydroxide solution; 500 c.c. of 
distilled water; 50 c.c. of alcohol; 50 c.c. of kerosene oil; a dozen 
1 gram sodium chloride tablets; 4 grams of sodium carbonate and 
1 ounce of a 1 per cent, aqueous solution of alizarin. One suction 
pump should be available for every six men. On the day preceding 
the actual experiment supply every two students with two separate 
portions of 5 c.c. of oxalated blood, obtained from a case of advanced 
nephritis. The student should place each portion in a test-tube 
labelled A and B respectively. Tube A receives 25 mg. of urease- 
soy bean extract (Hynson, Westcott & Dunning tablets), which is 
preferably placed in the tube, pulverized and suspended in 2 or 3 
c.c. of water, before the blood is added. Both specimens are then 
layered with 0.5 to 1 c.c. of toluene, well shaken, tightly corked and 
kept overnight at room temperature. 

Directions to the Student. — Transfer the contents of tube A to 
cylinder A by the aid of a few cubic centimeters of water, not more 
than five; add an equal quantity of alcohol, 2 grams of sodium 
chloride — two 1-gram tablets which should first be broken into 
small pieces, and a layer of kerosene oil, about 2 c.c. The contents 
of tube B are placed in cylinder B and treated like A. 25 c.c. of 3 \ 
sulphuric acid are placed in each one of the two Erlenmeyer flasks, 
with which you have been provided, and diluted with a similar 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 45 



quantity of distilled water. 0.5 to 1 gram — a good kuifepointful 
— of sodium carbonate is finally added to each cylinder, when 
cylinders and flasks are tightly closed with their respective stoppers, 
united with each other and connected with a suction piunp, as shown 
in the illustration (eliminating the flask at the extreme left). All 
three sets of apparatus from your group are connected with the same 
pump, the absorption bottle D being placed at the extreme right end. 
An air current is now passed through the entire system for a period 
of one hour. At the end of this time the current is stopped, the 
groups of apparatus and their individual components are discon- 
nected, and the contents of flasks A and B titrated with ^ alkali 
to the violet point, using four drops of alizariu as indicator. 
Deduct the amount of ammonia evolved in B from A. You 
will then find how many cubic centimeters of the 25 of ^ acid 
were neutralized by the ammonia resulting from the decomposi- 
tion of the urea contained in the quantity of blood with which 
you started. From this calculate the amount of urea per 1000 c.c, 
of blood, bearing in mind that 0.6 mgm. of urea will give rise to a 
quantity of ammonia corresponding to 1 c.c. of ^ acid. When 
noting your results show exactly, step by step, burette readings 
included, how you arrived at your final figure. 

Answer the Following Questions in Writing. — How much urea is 
normally found in the blood, per liter? How much would you expect 
to find in the case of a patient whose kidneys are more or less diseased? 
What prognostic conclusion would you draw from the quantity 
found? Write the equation which expresses the transformation of 
urea into ammonium carbonate, as the result of the action of the 
urease. Why do you add sodium carbonate to the mixture, in each 
cylinder? Why do you deduct value B from value A? What do 
you mean by the non-protein nitrogen of the blood ? Name the three 
most important substances belonging to this order? Retention of 
which one of the three is most apt to occur first, in the course of a 
developing nephritis? Which next and which last? What are the 
fluctuations in quantity of the non-protein nitrogen in the absence 
of renal disease? What percentage of the total is represented by 
the three most important components, individually? What values 
would you expect in a case of chronic nephritis associated with 
hypertension? Under what conditions would you expect a material 
increase in the amount of the so-called free ammonia ? 

Reading. — 
5 



46 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

Lesson 18. — The Microscopic Examination of Transudates 
and Exudates. — Material Required. — Smears on slides of the 
cellular components (a) of a typical transudate ; (6) of a transudate 
obtained from a case of peritoneal carcinomatosis; (c) of an exudate 
obtained from a case of tubercular pleurisy or peritonitis ; (d) of an 
exudate obtained from a pyogenic peritonitis; (e) of pus from a case 
of empyema; (f) of pus from a case of gonorrheal urethritis. The 
smears a to d should be fixed by heat and stained with a 1 per cent, 
aqueous solution of methylene-blue or with carbol thionin; e to f 
should be stained with Jenner's stain. 

Directions to the Student. — In specimen (a) note the comparative 
scarcity of the cells and the types — endothelial cells and small 
mononuclear leukocytes; draw these types and differentiate 50 
cells; give percentage values. In specimen (6) note the types — 
endothelial cells, tumor cells, showing evidence of atypical mitosis, 
leukocytes; note the large number of cells in general and of cells 
presenting an epithelial habitus; differentiate 100 cells and give 
percentage values. Draw the different types. In specimen (c) 
note the number of cells and the types — leukocytes and endothelial 
cells; differentiate 100 cells and give percentage values. Sketch a 
representative field. In specimen (d) note the number of cells; 
differentiate 100 cells and give percentage values; note the number 
and types of bacteria and the occurrence of phagocytosis on the part 
of many of the cells. Sketch a representative field. In specimen (e) 
note the same factors as in (d) ; observe also the absence or scarcity 
of eosinophiles; differentiate 100 cells and give percentage values. 
Sketch a representative field. Study specimen (/) in the same 
manner as (e); note the presence of eosinophiles and epithelial 
cells; study the morphological appearance of the gonococci and their 
occurrence within the polymorphonuclear neutrophiles; differen- 
tiate 100 cells and give percentage values. Sketch a representative 
field. 

Answer the Following Questions in Writing. — What is the essential 
difference between a transudate and an exudate? What is meant 
by the term "cytological study" of the various effusions? Generally 
speaking, how would you distinguish on cytological grounds, be- 
tween an exudate of tubercular origin and one due to pyogenic 
organisms ? Does a lymphocytosis invariably indicate tuberculosis ? 
What influence does chronicity of the pathological process which 
gives rise to the formation of an effusion, have upon the character 
of the cytological findings? What organisms are most apt to be 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 47 

encountered in exudates? In a suspected case of tubercular effusion, 
what examination, aside from a microscopic one of the cellular 
elements, would suggest itself to you as advisable? How would 
you proceed? What would you expect to find in a positive case? 
^Yhat is meant by a chylous exudate? Under what pathological 
conditions may such be found ? To what is the chylous appearance 
due? 

Reading. — ■ 

Lesson 19. — Material, Apparatus and Reagents Required. — Fur- 
nish a tubercular specimen of sputum, in Board of Health vials, 
to every six men; so, also, approximately, 10 c.c. of antiformin and 
an equal amount of a mixture composed of 1 part of chloroform and 
9 parts of alcohol. Supply every two students with 1 or 2 oz. of a 
1 per cent, aqueous solution of methylene-blue; of Jenner's stain; 
of carbol fuchsin and Gabbett's methylene-blue-sulphuric acid 
mixture. Staining trays and wash bottles, filled with ordinary tap 
water, should also be available, besides Bunsen burners, slides, 
nichrome wires and individual paper napkins. Provide basins 
containing 10 per cent, lysol and an Arnold sterilizer for disin- 
fecting purposes. 

Directions to the Student. — Each student should prepare two fairly 
thin smears from the most purulent portion of the sputum and one 
or two thicker smears and mark them (a), (b) and (c) respectively. 
The specimens should either be allowed to dry in the air or by 
"beating" the flame of the Bunsen burner — touching the hand 
after every few beatings, so as to prevent overheating. After being 
dried in this manner stain specimen (a) for a minute or so with the 
aqueous methylene-blue solution; lay the slides upon the carrying 
rods of the tray and flood them with the stain; wash off with water, 
cleanse the reverse side with a paper napkin and dry the smear by 
beating the Bunsen flame. Stain {b) with Jenner's stain in the same 
manner as you would stain a blood smear; do not dry by heat after 
staining. To stain specimen (c) raise up the carrying frame from 
the staining tray and set it obliquely across the latter; lay your 
slide across the rods, flood it with carbol fuchsin solution, bring this 
to steaming by heating with the Bunsen flame; keep the solution at 
the steaming point for a couple of minutes, adding more stain, if 
need be, to keep the smear well covered; wash off with water; flood 
the slide with the acid-methylene-blue mixture; leave this for three 
or four minutes; wash off with water; cleanse the reverse side of 



48 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



the slide with a paper napkin; dry the smear by beating the Bunsen 
flame. 

To the remainder of the sputum in the vial, add approximately 
one-fourth the quantity of antiformin and allow the mixture to 
stand at room temperature, well stoppered, until the next day, or 
in the incubator at 37° to 40° C. for one hour, shaking from time to 
time. After the mixture has become homogeneous, add for every 
10 c.c. approximately 1.5 c.c. of the solution composed of 1 part of 
chloroform and 9 parts of alcohol; shake. Pour the mixture into 
centrifugalizing tubes — one for every two men of your group of six; 
centrifugalize for fifteen minutes; pour the supernatant fluid into 
the lysol basin, make smears from the residue ; fix by heat and stain 
with carbol fuchsin and acid methylene-blue, as directed above; 
mark these slides (d) . Place a drop of immersion oil upon each one 
of your smears and examine with the oil-immersion lens. 

Note in (a) that the cellular elements are for the most part 
polymorphonuclear or polynuclear leukocytes, but that small 
mononuclear leukocytes also are present; search for round or oval 
" alveolar" epithelial cells and for irregular squamous epithelial 
cells derived from the pharyngeal district; note the large number of 
bacteria and the predominance of cocci, some in diplo, others in 
chain form and still others occurring singly or in groups; cocci 
predominate over bacilli. Sketch a representative field. In the 
Jenner specimen (6) note that among the leukocytes there is an 
occasional eosinophile; look also for the presence of red cells. In 
specimen (c) you will observe that the various cellular elements and 
bacteria which you noticed in (a) and (b) are not so sharply brought 
out. The blue here merely serves as a background stain to facili- 
tate the finding of any tubercle bacilli that may be present. The 
latter are colored red. Note their size, their frequent arrangement 
in groups of two or three, or in clumps of larger numbers; look for 
' 'beaded" specimens and organisms united so as to resemble in form 
the letter S. Make a composite sketch of your findings. In specimen 
(d) the cellular elements and the other organisms seen in (a), (b) 
and (c) will be missed; they have been destroyed by the antiformin; 
the tubercle bacilli will be found to be more numerous, as a result 
of the concentration of the sputum. At the end of the lesson place 
all sputum containers and soiled material in the steam sterilizer and 
run this for thirty minutes. 

Answer the Following Questions in Writing. — What elements give 
rise to the opaque appearance of the sputum ? What is the probable 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 49 



origin of the greenish color of the sputum? What epithelial ele- 
ments are likely to be found in sputum ? What cell is the common 
pus corpuscle ? What kinds of bacteria, besides the tubercle bacillus, 
would you expect to find in sputa coming from a tuberculous patient ? 
In what respect does the tubercle bacillus differ from most of the 
associated organisms found in sputa in reference to its behavior 
toward the usual bacterial stains, when used in aqueous or alcoholic 
solution? What is meant by a mordant? Name the two mordants 
which are most commonly employed in staining the tubercle bacillus ? 
What role does the sulphuric acid in Gabbett's stain play in the 
demonstration of the tubercle bacillus? Explain the term acid-fast. 
Name other bacilli that are acid-fast and where are they found? 
Of what significance is the presence of elastic tissue in sputum? 
Reading. — 

Lesson 20. — Material, Apparatus and Reagents Required. — For 
every six students supply the following: A specimen of sputum 
from a case of pneumococcus pneumonia; a couple of old forceps; 
a large basin with 10 per cent, lysol solution ; half a dozen tubes of 
meat infusion broth, 0.3 to 0.5 per cent, acid to phenolphthalein, 5 
c.c. to the tube; 5 c.c. of a sterile 20 per cent, solution of dextrose; 
5 c.c. of sterile, defibrinated human or rabbit blood; wash bottles 
with water. 

Furnish every two students with a couple of sterile tumblers 
with sterile saline — closed w T ith paper caps and rubber bands; 
two sterilized capillary pipettes in guard tubes; one rubber nipple; 
1 oz. each of Jenner's stain, Sterling's gentian- violet-anilin stain, 
formalin, Gram's iodin solution, 94 or 95 per cent, alcohol, carbol- 
fuchsin (diluted 1 to 10); dilute gentian- violet solution — 5 c.c. of a 
saturated alcoholic solution plus 95 c.c. of distilled water; 100 c.c. 
of a 20 per cent, aqueous solution of copper sulphate. Supply also 
a steam sterilizer for final sterilization of the sputum containers 
and other contaminated apparatus. Bunsen burners should be 
ready for use. 

Directions to the Student. — Examine the patient's sputum with the 
naked eye; note its rusty appearance in places and its viscidity; 
the container can frequently be inverted without spilling a drop of 
the contents. With the forceps supplied, separate a bit of the 
sputum from the main mass and prepare four smears on slides, 
using your nichrome wire or a tooth-pick, which latter should be 
burned immediately after use. Dry the specimens by beating the 



50 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

Bunsen flame and mark them a, b, c and d. Before proceeding to 
stain these, every student should add to one tube of the meat 
infusion broth 0.25 c.c, approximately 4 or 5 drops, of both the 
sterile dextrose solution and the defibrinated blood that has been 
supplied to his group. Then transfer a piece of the sputum, about 
the size of a bean, to one of the tumblers containing sterile saline; 
wash it well by floating it about; transfer it to the second tumbler; 
repeat the washing there and finally place it in the culture tube 
containing the dextrose-blood-meat infusion broth (Avery's so- 
called " artificial mouse")- Label the test-tube with your name and 
date and place it in the incubator — in a tumbler with some cotton 
at the bottom, to prevent breakage. Remove it from the incubator 
the following morning and examine the culture in the afternoon — 
in actual practice five to six hours incubation is sufficient. Now 
return to your sputum smears and proceed as follows: Stain (a) 
with Jenner's stain for about three minutes; wash off; allow to 
dry in the air. Stain (b) for one-half to one minute with Sterling's 
gentian- violet-anilin stain; pour off the excess of stain and cover 
the slide with Gram's iodin solution; after two or three minutes 
pour off the iodin solution and replace it with two or three changes of 
alcohol, — two to three minutes, until no more stain can be extracted 
in this manner; counterstain with dilute carbol-fuchsin solution, 
wash off, dry by blotting or beating the Bunsen flame. Flood 
(c) with formalin for three to five minutes; wash off with water; 
stain with Sterling's solution for one-half to one minute and con- 
tinue as with specimen (6). Stain specimen (d) for a few seconds 
with the dilute gentian- violet solution, which has been thus marked, 
staining the preparation for a few seconds; wash off with 20 per 
cent, copper sulphate solution; blot (do not wash). Examine all 
four specimens with the oil-immersion lens. In specimen (a) note the 
character of the cellular elements and the presence of large numbers 
of end-to-end diplococci, which often appear surrounded by a little 
colorless halo. In specimen (b) you will observe that the organisms 
have retained the gentian- violet stain and are hence Gram-positive. 
The treatment of (c) and id) was intended to bring out the presence 
of capsules; the methods employed were those of Wadsworth and 
Hiss respectively. With the former the capsule appears colorless; 
with the latter a faint blue. The organisms in question are pneumo- 
cocci. Draw representative fields. 

Answer the Following Questions in Writing. — What organism is 
the most constant cause of acute croupous or lobar pneumonia? 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 51 



To what is the rusty color of pneumonic sputum due? What are 
the essential morphological characteristics of the pneumococcus ? 
Is it a hemolyzing organism ? Are pneumococci found in the throats 
of healthy individuals who have not been in contact with pneumonia 
cases. What is the essential difference between such organisms 
and those found in the sputum of the majority of pneumonia 
patients? What is meant by type pneumococci? How many types 
are there? What percentage of pneumonia cases are due to these 
types individually? What is the corresponding mortality? Why 
is it necessary to wash the sputum before culturing it? What is the 
object of typing pneumococci in concrete cases? How would you 
distinguish between streptococci occurring in diplo-form and pneu- 
mococci, aside from the presence or absence of a capsule? 
Reading. — 

Lesson 21. — Material, Apparatus and Reagents Required. — The 
cultures made in Avery's medium, on the day preceding, should be 
removed from the incubator in the morning and kept in the refriger- 
ator or at room temperature until the class meets in the afternoon. 
For every six men provide the following: 10 c.c. of antipneumococcus 
serum, corresponding to the three types, diluted according to their 
titer; 5 c.c. of sterile ox bile; a basin containing 10 per cent, lysol. 

Every man individually should be furnished with a dozen steril- 
ized agglutination tubes, closed with cotton stoppers; corresponding 
racks; half a dozen sterile capillary pipettes in guard tubes, with 
a couple of nipples; one short sterile test-tube of ordinary size; a 
wax pencil. 

. Supply also a steam sterilizer to disinfect contaminated material 
at the end of the lesson; an ordinary incubator kept at 37° C, 
and a high-speed centrifuge. In addition supply the same reagents, 
used for capsule staining, that were employed the day before. 

Directions to the Student. — Prepare a smear from your culture, 
using your nichrome wire loop; dry and stain it according to Hiss's 
or Wadsworth's method (see preceding lesson). Examine with the 
oil-immersion lens. If the culture is a fairly pure one, of capsulated 
diplococci, centrifugalize for a couple of minutes at low speed to 
throw down the red cells. Transfer the supernatant fluid to a 
sterile test-tube, using one of your capillary pipettes. 

Experiment A {Agglutination Test). — Charge three agglutination 
tubes marked I, II and III, respectively with approximately 0.5 c.c, 
8 drops, of the culture, and add a corresponding quantity of the 



52 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



respective antisera, I, II and III, taking care to thoroughly mix 
the two components. Place the tubes in the incubator at 37° C. 
and examine them at intervals of half an hour. If the diplococcus in 
question was a type organism agglutination, visible with the naked 
eye, will be noticeable after one to two hours' standing. In any 
case reexamine the next day, meanwhile allowing the tubes to stand 
at room temperature. If the organism is not a type organism, but 
belongs to group IV, the tubes will appear equally turbid and no 
clearing will have taken place. Sketch the final result. 

Experiment B (Precipitin Test). — While the tubes charged, as 
directed above, are in the incubator, add 1 c.c. of sterile ox bile to 
the remainder of your culture, and incubate for twenty minutes. 
If the culture was pure the turbidity will clear up owing to the solv- 
ent action of the bile upon the pneumococci. If the turbidity has 
not cleared up entirely, centrifugalize at high speed, until the fluid 
is perfectly clear. Now charge three agglutination tubes marked 
I, II and III with 0.5 c.c, 8 drops, of the clear fluid, and add a similar 
quantity of the corresponding antisera as directed in experiment A. 
The results may be read in a few minutes and are sharp and dis- 
tinct; the occurrence of a turbidity in one of the tubes indicates that 
you are dealing with a type organism, the number of the type 
corresponding to the number of the antiserum. No turbidity will 
develop, if the organism in question belongs to group IV. Sketch 
the final result. Sterilize all contaminated material in the steam 
sterilizer, at the end of the lesson. 

Answer the Folloiving Questions in Writing. — What is the 
technical term that is applied to the substance in the antiserum 
which causes the agglutination of the corresponding organisms? 
What is meant by the term precipitin? What does it precipi- 
tate? What is the effect of bile or bile salts upon pneumococci? 
How would you secure a specimen of sputum, actually coming 
from the lungs, in a case of pneumonia? If you had reason 
to believe that your specimen was contaminated by mouth organ- 
isms, but that organisms from the lung were also present, how would 
you proceed ? In what other manner could you secure a representa- 
tive culture, if sputum should not be available? Having determined 
in a given case, to which group the organism belongs, what would 
be the corresponding prognosis? What indication for treatment 
would the examination afford? 

Reading. — 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 53 



Lesson 22. — Material, Apparatus and Reagents Required. — For 
every six students provide 2 oz. of the following stains: a freshly 
prepared mixture of Neisser's methylene-blue and Neisser's crystal 
violet, 2 parts of the first and 1 of the latter; Neisser's chrysoidin or 
Bismarck-brown solution; Loeffier's blue solution; Sterling's gentian 
violet-anilin solution; Gram's iodin solution; 94 per cent, alcohol; 
carbol fuchsin solution and Hiss's capsule stain solutions (see Lesson 
20); cultures of the diphtheria bacillus on Loeffier's medium; of the 
Streptococcus hemolyticus, Streptococcus viridans and the pneu- 
mococcus on human blood-agar plates and of the meningococcus 
on a serum plate; 20 c.c. of a sterile, rich emulsion of human blood 
in dextrose broth. 

Furnish every student with three small sterilized cotton 
swabs, two on straight reeds, one on a wire bent near the end to an 
angle of about 145°; a wooden tongue depressor; two tubes of 
Loeffier's blood serum; one tube of about 10 to 15 c.c. of agar; one 
sterile capillary pipette with nipple; one sterile Petri dish; one plate 
of dextrose serum agar, 0.4 per cent acid to phenolphthalein; a 
nichrome wire and a wax pencil. Have available also staining trays, 
paper napkins, tumblers, cotton, an incubator at 37° C, basins con- 
taining 10 per cent, lysol solution; a steam sterilizer to disinfect all 
contaminated material at the end of the lesson. 

Directions to the Student. — Put approximately 3 c.c. of the emul- 
sion of human blood in your Petri dish, melt the tube of agar, 
furnished you, by heating in the free flame of the Bunsen burner; 
be careful not to crack the tube; when entirely liquefied, allow 
it to cool for a few minutes, until you can just handle the 
heated portion of the tube; flame the mouth of the tube and 
pour the contents into the Petri dish containing the blood; 
mix the two well by circular rotation; then set aside to harden 
for half an hour. While this is taking place study the appearance 
of the cultures furnished your group, with the naked eye; compare 
the different types; make a sketch illustrating the difference in the 
type of hemolysis, shown by the two varieties of the streptococcus 
and the pneumococcus. Note (a) the narrow greenish zone of dis- 
coloration of the latter; (b) the green color and absence of hemol- 
ysis of the viridans colonies; (c) the distinct hemolytic area, meas- 
uring 2 or 3 mm., surrounding those of the Streptococcus hemo- 
lyticus. Now make two smears of each one of the different organ- 
isms, in drops of water; dry them by beating the Bunsen flame, and 
mark them Da and Db, Va and Vb, Ha and Hb, Ma and 1Mb, Pa and 
6 V 



54 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



Pb (D = B. diphtherise, V = S. viridans, H = S. hemolyticus, 
M = meningococcus, P = pneumococcus) . Stain Da for ten to 
fifteen seconds with the Neisser blue mixture; wash off; counter- 
stain for an equal length of time with Neisser's chrysoidin solution 
or with Bismarck brown ; wash off ; dry the reverse side with a paper 
napkin; then beat the flame, until dry. Stain Db for five to ten 
minutes with Loeffler's blue; wash off and dry as just directed. Stain 
Va, Ha, Ma and Pa according to Gram, and Vb, Hb, Mb and Pb 
with Hiss's capsule stain, as directed in Lesson 20. Now examine 
each slide with the oil-immersion lens - and sketch representative 
groups of the organisms, indicating what each sketch is intended to 
show. In Da note particularly the polar bodies and their arrange- 
ment; in Db the form of the organisms. Compare the form and 
absence of capsules in Va, Vb, Ha and Hb, Ma and Mb, as compared 
with Pa and Pb. Note that the streptococci and the pnemococci 
are "end-to-end" organisms and Gram-positive, whereas the menin- 
gococci show a " side-to-side" grouping and are Gram-negative. 
After completing your microscopic work, make cultures from your 
working partner's throat and nose on the culture media furnished as 
follows: Hold down the tongue with the wooden depressor; apply 
one of the straight reed-swabs to the tonsils, if possible, gently 
entering the large anterior crypt, and now smear one of the Loeffler 
medium slants; mark this T. Take a second reed-swab and enter 
the nose, directing the reed straight backward, not upward; gently 
press it back to the posterior portion and now smear the second 
Loeffler slant with the material obtained; mark the tube N. 

With your third swab attempt to reach the nasopharynx, behind 
the velum of the palate and then smear both the serum-agar plate 
and the blood-agar plate; mark both NP and with the name of the 
subject. Set your tubes in a tumbler, on cotton, and mark the 
tumbler with your partner's name. Incubate both tubes and plates 
at 37° C. until the following afternoon, when they are examined as 
directed in Lesson 23. 

Answer the Following Questions in Writing. — Are diphtheria 
bacilli only found in the throat of patients suffering from diphtheria, 
or convalescents from the disease? What is meant by active and 
what by passive carriers? Why should an examination of the nose 
of an individual who has recently passed through the disease or who 
has been in contact with the malady be invariably made, in addition 
to an examination of the throat, before concluding that he is not 
a carrier? How would you determine whether the organisms are 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 55 



virulent ? If you should meet with a patient presenting a suspicious- 
looking exudate in his throat, how could you secure a satisfactory 
culture if the usual media should not be at hand? What other 
organisms, besides the diphtheria bacillus, may give rise to the 
formation of exudates about the tonsils? What is Vincent's angina 
and to what organism is it due? Name the most important lesions 
which are produced by the Streptococcus viridans and the Strepto- 
coccus hemolyticus respectively? What type of streptococcus is 
usually associated with septic sore-throat? Are any of these organ- 
isms ever met with in the throats of individuals who are, at the time, 
not suffering from the corresponding infections; is there any ground 
for the belief that such individuals may act as carriers? What 
infections seem to predispose to the pathogenic activity of the 
Streptococcus hemolyticus? Given an outbreak of streptococcus 
infection, in the surgical division of a hospital, how would you pro- 
ceed to trace its origin and to prevent its further spread ? 
Reading. — 

Lesson 23. — Material, Apparatus and Reagents Required. — The 
tubes and plates inoculated by the students from each other's 
throats and noses, during the preceding lesson, after twelve to 
twenty-four hours' incubation, 

For every six students furnish also a living meningococcus cul- 
ture and the same stains as in the preceding lesson. 

Supply each student with one tube of veal infusion broth, one 
tube of Hiss's serum water inulin and four agglutination tubes, each 
containing 1 c.c. of Olitsky's medium. 

Have available lysol basins and a steam sterilizer. 

Directions to the Student. — Examine your cultures on the Loeffier 
medium for diphtheria bacilli, using Xeisser's stain. If none are 
found, stain smears with dilute carbol fuchsin, for a minute or so, 
and try to ascertain the character of the organisms that have grown 
out, on the basis of their morphology. Sketch what you see. Then 
examine your blood -agar plates, with the naked eye, for the presence 
of greenish or hemolyzing colonies; fish individual colonies, make 
smears and stain (a) according to Gram, using Sterling's gentian- 
violet anilin solution, and (6) for capsules, using Hiss's method. 
Having identified colonies of the Streptococcus hemolyticus, so far 
as this is possible by microscopic methods, inoculate a tube of veal 
infusion broth with a carefully fished colony; mark it and incubate 
for twenty-four hours; examine its hemolytic action the next day in 



56 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



the test-tube experiment and ascertain whether the organism is 
bile-soluble or not. Sketch the microscopic appearance of the organ- 
isms which you regard as streptococci. Inoculate one tube of Hiss's 
serum water — inulin medium with a colony; observe this daily for 
three or four days. Now examine your serum-agar plate, at first 
macroscopically; then fish colonies and see whether any of them are 
composed of Gram-negative organisms, and present a " side-to-side" 
grouping. If so, fish suspicious-looking colonies from the plate and 
transfer to tubes of Olitsky's medium, one colony to a tube; mark 
the tubes and the corresponding colonies on your plate and incubate 
overnight. Finally, inoculate one tube of Olitsky's medium with a 
loopful of living meningococci to serve as a positive control, and 
place this in the incubator. 

Answer the Following Questions in Writing. — What is the action 
of bile or bile salts on streptococci? what is it upon pneumococci? 
What is the action of streptococci upon Hiss's serum water inulin 
medium as compared with that of pneumococci? Is chain formation 
typical of streptococci? What is the principle underlying Olitsky's 
method of identifying the meningococcus? What is meant by the 
normal horse-serum negatives? Name those which are likely to be 
encountered in the nasopharynx. What is the behavior of the 
horse-serum negatives toward staining by Gram? With what 
organism, aside from the horse-serum negatives, is the meningo- 
coccus most likely to be confused? How do you finally distinguish 
between the two when using Olitsky's method? 

Reading. — 

Lesson 24. — Material, Apparatus and Reagents Required. — The 
cultures made during the preceding lesson. 

For every six men provide 20 c.c. of a freshly prepared 5 per cent, 
emulsion of washed sheep cells; 2 c.c. of sterile ox bile or 2.5 c.c. of 
a sterile 10 per cent, solution of sodium taurocholate; 2 c.c. of a 
sterile 10 per cent, dilution of a polyvalent antimeningococcus 
serum, of high titer, in 0.85 per cent, saline. 

Furnish every student with three or four sterile plugged agglutina- 
tion tubes, the same number of sterile capillary pipettes and a 
couple of rubber nipples. Provide lysol basins and steam sterilizers. 

Directions to the Student. — Transfer 1 c.c. of your streptococcus 
culture, in veal infusion broth, to one of your agglutination tubes, 
and 1 c.c. to a second tube; sterilize your pipette by burning it in the 
flame of the Bunsen burner. Mark your tubes H and B respectively. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 57 



To H add an equal volume of the 5 per cent, emulsion of sheep cells, 
supplied to your group; shake well and incubate for two hours at 
37° C, centrifugalize and note whether hemolysis has occurred or 
not. Sketch the final result, using your red pencil to indicate the 
occurrence and degree of hemolysis. To B add three drops of ox 
bile or three drops of the sodium taurocholate solution; sterilize 
your pipette by heat; mix well and incubate for half an hour at 
37° C. Sketch the final result and illustrate at the same time what 
would have happened if your culture had been one of the pneumo- 
coccus. Now study the results which you obtained with your 
cultures made from the veal infusion agar plate. Set the horse 
serum negatives, i. e., those tubes in which agglutination has taken 
place, aside and add to those which are turbid one drop of the anti- 
meningococcus serum supplied to your group. Incubate in a water 
incubator for two hours and finally make a sketch of your results. 

Answer the Following Questions in Writing. — In what portion of 
the upper respiratory tract is one most liable to encounter meningo- 
cocci in carriers? How long does the carrier stage usually last? 
What is meant by a "polyvalent" antiserum? 

Reading. — 

Lesson 25. — Material, Apparatus and Reagents Required. — For 
every six men provide 4 c.c. of spinal fluid obtained from a case of 
paresis; the fluid must be free from blood; further, supply the various 
reagents used for the Wassermann test, in the quantities indicated 
in Lesson 13, for groups of six men; likewise the necessary glassware 
— a set for every two men; a water incubator at 37° C. and a 
few cubic centimeters of a neutral saturated solution of ammonium 
sulphate. 

For every two men also furnish 11 test-tubes of ordinary size, 
one 2 c.c. pipette, graduated in tenths; half a dozen capillary 
pipettes, with rubber nipples, and one 5 c.c. pipette — all of which 
should have been cleansed, as is usual with glassware that is to be 
used for the Lange colloidal gold test. This set is to be used only 
for "this purpose. In addition, furnish every six students with an 
ordinary test-tube rack; 25 c.c. of 0.4 per cent, saline made from 
triply distilled water and 100 c.c. of colloidal gold solution, both in 
adequately cleansed beakers. Furnish also individual blood-count- 
ing chambers. 

1. Directions to the Student (two men working together). — Take 
two Wassermann tubes and mark them A and B respectively. 



58 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



Charge both with 0.2 c.c. — 4 drops — of the spinal fluid, furnished 
your group; add to each one the same quantity of complement 
(which has already been diluted) ; A further receives 0.2 c.c. of the 
diluted antigen and B 0.2 c.c. of saline. Incubate the tubes in the 
water-bath, or your vest pocket for fifteen minutes; then add to 
each tube 0.2 c.c. of the sheep cell emulsion and 0.2 c.c. of the diluted 
amboceptor. Reincubate for fifteen minutes, centrifugalize and note 
the results. What does the A tube show? what does the B tube 
show? Is the reaction positive or negative? Sketch the appearances 
of the tubes. 

2. Set up the eleven test-tubes, furnished for the colloidal gold 
test, and mark them 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. Using your 
2 c.c. pipette, charge tube 1 with 1.8 c.c. of the 0.4 per cent, saline 
and the remaining tubes with 1 c.c. each. Now add 0.2 c.c. — 4 
drops — of spinal fluid to tube 1, using a capillary pipette, and break- 
ing this off at a point, so as to furnish a fairly large drop. Take up 
your 2 c.c. pipette again, blow bubbles into tube 1, so as to thoroughly 
mix the fluid with the saline; transfer 1 c.c. to tube 2; blow bubbles; 
transfer 1 c.c. from tube 2 to tube 3, and so on, until tube 10 has been 
deprived of 1 c.c. of its mixture; this 1 c.c. is discarded. Tube 11 
thus only contains saline and serves as a color control in the end. 
Now add 5 c.c. of the gold solution to each one of the 11 tubes; 
shake each tube aud set the entire lot aside until the end of the period 
when they should be examined and the color in each noted. Let the 
tubes stand over night and reexamine them the next day. The 
appearance of a bluish tint is noted as 1, lilac as 2 and dark blue as 
3, a pale blue as 4 and decolorization as 5, while no change in color 
is denoted by the mark 0. Express your readings accordingly, in a 
number made up of 10 numerals. Sketch the result, using colored 
pencils. 

3. Directions to the Individual Student. — Place 5 drops of the satur- 
ated solution of ammonium sulphate, supplied to your group, in 
one agglutination tube, add 5 drops of spinal fluid, and note whether 
a turbid ring appears at the zoue of contact of the two fluids, either 
immediately or on standing for a few minutes (Ross-Jones test). 
Normal fl.uid will not give a turbidity. Sketch the result. 

Mount a drop of the undiluted spinal fluid on the platform of your 
blood counting chamber; adjust the cover-glass; let stand for a few 
minutes; then count the number of leukocytes in 100 large squares 
using the middle power and taking care not to confound red cells 
that may be present with leukocytes. Try to ascertain whether the 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 59 



leukocytes in question are polynuclears or mononuclears; calculate 
the number for 1 c.mm. of the fluid and give this in your report. 

Answer the Following Questions in Writing. — What is the normal 
cell count of the cerebrospinal fluid? Under what pathological 
conditions would you expect an increase? What does the Ross- 
Jones reaction indicate? Under what pathological conditions is it 
obtained? What is meant by the term a "colloidal" solution of 
gold? What happens to this solution when a change in color tend- 
ing toward blue occurs upon mixing it with diluted cerebrospinal 
fluid — in a certain concentration — obtained under certain patho- 
logical conditions? Why is it necessary to use doubly or triply 
distilled water in making up the reagents, and why is it necessary 
to cleanse the glassware so thoroughly before use? What consti- 
tutes the "paretic curve?" Under what pathological conditions 
would you expect a positive Wassermann in the cerebrospinal fluid ? 

Reading. — 

Lesson 26. — Material, Glassware and Reagents Required. — Fur- 
nish every student with a cerebrospinal cell smear, obtained (a) 
from a normal individual, or at any rate from one who has no dis- 
ease of the meninges or the central nervous system ; (6) from a case 
of tabes; (c) from a case of paresis; (d) from a case of acute anterior 
poliomyelitis; (e) from a case of epidemic meningitis; (J) from one 
of tubercular meningitis; (g) from one of pneumonic meningitis- 
pneumococcus or streptococcus type. All specimens should be 
stained with methylene-blue. 

Furnish also a double set of unstained smears from a case of gonor- 
rheal urethritis, and the necessary reagents to stain by Gram — a 
set for every six men; supply staining trays and wash bottles. 

Directions to the Student. — Examine each slide, first with the low- 
power — to get a general idea of the number of cells present and their 
location; then with the oil-immersion lens; note the various types 
of cells that may be present — small mononuclear leukocytes, poly- 
morphonuclear neutrophiles and endothelial cells. In each slide 
ascertain the percentage number of the different types and note 
that in the pyogenic infections the polymorphonuclears predominate, 
while in the others the small monos control the picture. In the two 
cases of pyogenic meningitis search for the corresponding organisms 
— note their morphology and the occurrence or non-occurrence of 
phagocytosis. Sketch the various pictures and append appropriate 
legends. Now stain one of the smears of gonorrheal urethritis with 



60 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



the gentian-anilin solution, twenty to thirty seconds and the other 
according to Gram (see lesson). Then examine both specimens 
with the oil-immersion lens; note the morphology of the gonococci, 
the relative number contained within cells, as compared with those 
lying free, and the behavior toward Gram staining. Sketch the 
appearances. 

Answer the Following Questions in Writing. — Given a case of pyo- 
genic meningitis, how would you determine whether you are dealing 
with a meningococcus or a pneumococcus infection, aside from your 
microscopical findings? Given a case of suspected tubercular 
meningitis describe the technic which you would employ to ascertain 
whether this diagnosis is correct? 

Reading. — 

Lesson 27. — Material, Apparatus and Reagents Required. — (a) 
2000 c.c. of a normal digestive mixture, containing 0.365 per cent, of 
hydrochloric acid, half a teaspoonful of pepsin powder, and a couple 
of slices of bread; the mixture should be incubated for an hour at 
37° C. before class work begins; this mixture should be marked in 
such a manner as not to indicate its composition to the student. 
(6) 2000 c.c. of a 1 per cent, solution of lactic acid and a couple of 
slices of bread. This mixture also should be incubated for one hour 
and marked. For every six students provide 350 c.c. of the above 
two digestive mixtures; 200 c.c. of y~q sodium hydrate solution; 
25 to 30 c.c, each of a 0.5 per cent, alcoholic solution of dimethyl- 
amino-azo-benzol; 25 to 30 c.c. of a 1 per cent, aqueous solution of 
alizarin-mono-sulphonate of sodium; 25 to 30 c.c. of a 1 per cent, 
alcoholic solution of phenolphthalein ; 25 to 30 c.c. of a saturated 
aqueous solution of the sesquichloride of iron ; 25 to 30 c.c. of a con- 
centrated solution of carbolic acid; one eighth of the white of a hard 
boiled egg; 100 c.c. of milk. 

Every student should be furnished with: a Mohr burette with 
stand; five whiskey glasses or beakers of corresponding size; a 
stirring rod; half a dozen ordinary test-tubes and a 5 c.c. pipette. 

Directions to the Student. — Provide yourself with 50 c.c. each of 
the two digestive mixtures; mark them to correspond to the stock 
mixtures furnished your group; fill your burette to the 30 c.c. mark 
with T n o sodium hydrate solution; be sure that there are no bubbles 
in the rubber cuff. Measure off 5 c.c. of mixture A — unfiltered — into 
each one of your three whiskey glasses and mark these P, A and D 
respectively. To P add one or two drops of the phenolphthalein 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 61 



solution as indicator; to A three or four drops of the alizarin solution 
and to D three or four drops of the dimethyl-amino-azo-benzol 
solution. Take your burette reading; lower meniscus. Place a 
sheet of white paper beneath the burette and place the glass P upon 
this. While stirring with your glass rod allow the solution to 
flow into your mixture until a permanent pink color results; as soon 
as this point has been reached take your reading again; the number 
of cubic centimeters of the ^ alkali used, when multiplied by 20, 
will give you your total acidity. Then titrate A until a pure violet 
is obtained; the difference in your readings when multiplied by 20, 
will give you the acidity due to free acids and acid salts. Finally 
titrate D until the last trace of red has disappeared and the color 
has turned a greenish yellow; the difference in your readings, mul- 
tiplied by 20, will give you the acidity due to free hydrochloric acid. 
Note your findings in tabulary form, as shown below: 

Total acidity — 

Free HC1 . — 

Combined HC1 — 

Acid salts and organic acids — 

Next fill a test-tube with water; add one drop of the sesqui- 
chloride of iron solution ; mix the two and dilute still further, until 
the solution is almost colorless; pour one-half of the contents of the 
tube into a second tube and use one for comparison ; now add a few 
cubic centimeters of the digestive mixture to one of the tubes, and 
note whether or not a canary yellow color results — presence or 
absence of lactic acid (Kelling's test). To ascertain this point, 
test also, according to Uffelmann's method, without previous extrac- 
tion by means of ether: In a test-tube mix 3 drops of your chloride 
of iron solution with 3 drops of the concentrated carbolic acid solu- 
tion; dilute with water until an amethyst blue color results. To this 
mixture add a few cubic centimeters of the digestive mixture, when 
in the presence of lactic acid a canary yellow color will result. 

Now place approximately 15 c.c. of the digestive mixture in a 
test-tube; add a tiny slice of coagulated egg and incubate overnight. 
If pepsin is present the egg will be digested and undergo solution. 

Finally treat 5 to 10 c.c. of milk, in a test-tube, with 3 to 5 drops of 
the digestive mixture and incubate for ten to fifteen minutes at 37° 
C. If coagulation occurs during this time it may be inferred that 
the milk curdling ferment was present. Arrange your findings in 
tabulary form below your acidity determinations. After having 
completed the examination of the A mixture, repeat the entire proc- 



62 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

ess with the B mixture, and arrange your findings in a correspond- 
ing manner. 

Answer the Following Questions in Writing. — What is meant by 
free HQ? What by combined HQ? Define (a) euchlorhydria, (b) 
hyperchlorhydria, (c) anachlorhydria, (d) hyperacidity, (e) ana- 
cidity. What is meant by gastric hypersecretion? what by achylia 
gastrica? Under what pathological conditions is lactic acid in not- 
able quantity usually found? Under what pathological conditions 
does anachlorhydria occur? Why is it necessary to obtain the gas- 
tric contents at a certain time after the administration of a meal of 
known composition? What is the idea underlying the fractional 
analysis of the stomach contents? Describe in a general way the 
role which an indicator plays in volumetric analysis, using the titra- 
tion of a normal digestive mixture in the presence of dimethyl- 
amino-azo-benzol on the one hand and phenolphthalein on the 
other, as an example. 

Reading. — 

Lesson 28. — Material, Apparatus and Reagents Required. — For every 
six men furnish 2 c.c. of stomach contents, containing Boas-Oppler 
bacilli; a similar quantity of stomach contents containing sarcinse. 
The stomach contents should be procured the day before and allowed 
to stand in the ice-box overnight; the next morning the supernatant 
fluid should be carefully decanted from the sediment and the latter 
transferred to suitable little bottles and layered with toluol; thus 
preserved the material will keep for many months. Mark the bottles 
" Oppler-Boas bacilli" and "Sarcinae" respectively. 

For every six men furnish also small samples of moderately thin 
emulsions (a) of normal feces, (b) of a fatty stool, (c) of feces obtained 
from an acute enteritis or colitis. Also supply a somewhat larger 
sample — 5 to 10 grams — of a stool containing occult blood; mark 
this "to be examined for occult blood." 

Every six students should be furnished with 15 to 25 c.c. of alka- 
line phenolphthalin solution; a few cubic centimeters of a 10 per 
cent, solution of hydrogen peroxide; 0.5 gram of powdered benzidin; 
10 to 15 c.c. of glacial acetic acid; 10 c.c. of a 3 percent, solution of 
hydrogen peroxide; 2 oz. of a 1 per cent, aqueous solution of methy- 
lene-blue; 1 oz. of a saturated alcoholic solution (70 per cent.) of 
Sudan III; 1 oz. of Lugol's solution. 

Furnish every student with a few ordinary test-tubes, a small 
piece of filter paper, slides and cover-glasses. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 63" 



Directions to the Student. — First examine the fecal specimen 
marked "for occult blood," using (a) the phenolphthalin test and 
{b) the dry benzidin test, (a) In a test-tube emulsify a bit of the 
fecal material — the size of a pea — in 2 c.c. of distilled water — or 
at any rate in water which itself does not give the reaction, — add 
1 c.c, approximately, of the phenolphthalein solution and one or at 
most two drops of the 10 per cent, solution of hydrogen peroxide. 
In the presence of blood a pink color will develop, either at once or on 
standing for a few minutes, the rapidity of its appearance and its 
intensity varying with the quantity present. (6) Smear a tiny bit 
of fecal material — the size of a match-head — over a piece of white 
filter paper and drop on this 2 or 3 drops of benzidin solution. 
Prepare the latter yourself as follows: In a small test-tube place 
a little powdered benzidin, about twice the size of an ordinary pin- 
head; add 1 c.c. — 15 drops — of glacial acetic acid and 10 drops of a 
3 per cent, solution of hydrogen peroxide. In the presence of blood- 
coloring matter a greenishrblue color will develop on the filter paper 
in a few seconds. Next mount a droplet of the emulsified fecal 
material that you have been testing; adjust a cover-glass and exam- 
ine with the middle power for red blood cells. Xote that none can 
be demonstrated, the blood being occult. Now prepare a smear 
from the specimens of gastric contents, marked "Oppler-Boas 
bacilli" and ''Sarcina?" respectively; dry by beating the flame of 
the Bunsen burner and stain for thirty seconds with methylene- 
blue; wash off, dry and examine with the oil-immersion lens. The 
Oppler-Boas bacilli will appear as long, stout rods, occurring either 
individually or in chains. In the same specimen deeply stained 
yeast cells will also be found; look for budding forms. In the "Sar- 
cinar' specimen look for the characteristic cotton-bale like packets. 
Make sketches of your findings. Next moimt a small drop of the 
emulsified "normal" fecal material; adjust a cover-glass and examine 
with both middle and low power. Xote that the entire "back- 
ground" of the picture is made up of countless numbers of bacteria 
of various kinds, bacilli and cocci, either tmdergoing active or passive 
movement. With the low power search (a) for muscle fibers — these 
appear as yellow or yellowish-brownish cast-like formations, with 
parallel borders; with the higher power the transverse striation can 
usually be made out. Xote the relative number, bearing in mind 
that the stool is from a normal individual. With the low power 
search (b) for imdigested vegetable matter — spirals and cells which 
may or may not contain starch — the latter can be demonstrated 



64 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



by allowing a drop of Lugol's solution, added from the side, to mix 
with the specimen, when all starchy material will turn a bluish 
black; (c) look for vegetable hairs, presenting a central canal, either 
broken off or starting from a broad base; these are often mistaken 
for worms by the beginner. Draw these various appearances, and 
append corresponding legends. Now mount a drop of the specimen 
marked "fatty stool;" adjust a cover-glass and examine with the 
middle power. Note the enormous number of fatty acid crystals, 
occurring individually and in sheaves of various sizes; look also for 
fat globules. Allow a drop of the Sudan III solution to flow under 
the cover-glass, from the side, and note that the fat globules are 
colored by the dye. Draw. Finally, mount a drop of the "enteric" 
stool; adjust a cover-glass and examine with the middle power. 
Note the appearance of the admixed mucus and the adhering cells, 
which are, for the most part, desquamated columnar epithelial cells, 
characterized especially by the irregularity of their form; search 
also for pus corpuscles and red blood cells — note the small number 
of the former. Draw. 

Answer the Following Questions in Writing. — What organism is 
largely responsible for the production of lactic acid in the stomach 
contents of patients suffering from cancer of the stomach? Under 
what conditions are sarcinse liable to occur in the stomach contents? 
How is phenolphthalin formed from phenolphthalein ? What 
happens to the phenolphthalin when it is brought in contact with 
occult blood in the presence of hydrogen peroxide? Why does the 
solution turn pink? What happens to the benzidin under like condi- 
tions? What is meant by lientery; what by steatorrhea? Why will 
the latter condition result when the common bile duct is obstructed? 
Under what pathological conditions does mucus occur in the stool 
in large amounts? What is an enterolith? What is the most 
common bacterium occurring in the feces? 

Reading. — 

Lesson 29. — Material Required. — Furnish every student with a 
blood smear or blood smears showing (A) the parasite of benign 
tertian fever (a) in the early stage of its development, (b) forms 
twenty-four hours old, (c) mature forms; (B) the parasite of quartan 
fever, also if possible in various stages of its development; (C) the 
parasite of malignant tertian fever (a) in the early stage of its devel- 
opment, (b) presenting crescentic forms. Furnish also smears from 
the spleen or bone-marrow of a patient dead with malignant tertian 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 65 



fever to show adult schizonts. The specimens should all be stained 
with an eosin-methylene-azure mixture. 

Directions to the Student. — The smears should be studied with the 
oil-immersion lens; crescents may be searched for with the low power. 
Note that the body of the malarial parasite is colored blue while its 
chromatin appears red. In every cell determine whether the chrom- 
atin is present in a single compact mass or whether it shows evidence 
of division — a distributed nucleus. Note the melanin granules — 
their color and number — in the more mature organisms. Observe 
the occurrence of Schiiffner's dots — azurophilic granules — in the 
bodies of the red cells, harboring the parasite of benign tertian fever. 
Note that the red cells infected with plasmodium vivax tend to 
increase in size, while this is not the case in quartan and malignant 
tertian fever. Observe the signet-ring appearance of the young 
estivo-autumnal forms. Sketch representative cells showing these 
points and also the process of aging from the young to the adult 
form. In each one of your specimens search for male and female 
gametes; draw such cells and place alongside of them the corre- 
sponding schizonts. 

Finally, do a differential leukocyte count in one of your benign 
tertian, quartan and malignant tertian specimens, paying particular 
attention to the correct differentiation of the large mononuclears, 
as compared with the small mononuclear forms. 

Answer the Following Questions in Writing. — Why is an eosin- 
methylene azure mixture preferable to an eosin-methylene-blue 
mixture for staining the malarial parasite? To what class of pro- 
tozoa does the malarial organism belong? to what subclass? to what 
order? to what suborder? to what family? 

Write the Latin name of each one of the three types of the malarial 
organism that you have studied; give the generic and the species 
name. By whom was the malarial organism first discovered ? What 
is meant by schizogony? What term is applied to the products of 
schizogony? What is a schizont? W T hat is meant by sporogony? 
How are its products termed ? What is meant by gametes ? What 
is the difference between a gamete and a gametocyte ? What does 
the prefix macro and micro denote in connection with the term 
gamete or gametocyte? What is meant by the term zygote? How 
many hours does the benign and malignant tertian parasite require 
for its cycle of development? What is the most striking point in the 
leukocytic formula of a case of malarial fever? Prepare a table show- 
ing the differences between the three types of organisms. 

Reading. — 



66 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



Lesson 30. — Material Required. — Furnish each student (a) with 
a blood smear from a rat that has been infected with Trypanosoma 
lewisi or T. gambiense; (b) with a smear from a culture of Leish- 
mania infantum; both should be stained with an eosin-methylene- 
azure mixture; (c) furnish every six men with specimens of Anopheles 
maculipennis, Culex pipiens, Glossina palpalis, Pulex irritans, 
Cimex rotundatus, Ornithodorus moubata, Conorhinus megistus. 

Directions to the Student. — In specimen (a) note the morphology 
of the organism. The posterior end is drawn out in a long, pointed, 
nose-like projection. The large nucleus is located in the anterior 
third of the body. The blepharoplast is rod-shaped and stands at 
an angle to the body of the parasite, the flagellum originates near 
the latter; it runs forward in the margin of the undulating membrane 
and then continues by itself. Sketch these various points. Next 
examine specimen (6). Search for non-flagellated and flagellated 
forms; the latter develop from the former; note the general mor- 
phology, the presence of a macronucleus and a blepharoplast. The 
latter is smaller than the nucleus; it stains more deeply and is placed 
near the periphery. There is a single flagellum and no undulating 
membrane. Sketch these various features. Finally examine the 
various disease-carrying insects which have been furnished your 
group. Draw the wing of the two types of mosquitoes. 

Answer the Folloiving Questions in Writing. — To what class of 
protozoa do the trypanosomes and Leishmania belong? To what 
subclass? What pathological conditions in men are caused by the 
former and what by the latter? By what insects are these various 
infections carried ? Arrange your answers in tabulary form : Name of 
disease; causative parasite; transmitting insect. Make a diagram- 
matic sketch of the life-cycle of the malarial organism. By what 
organism is relapsing fever produced ? By what insect is the disease 
transmitted? Name the various spirochetes which are pathogenic 
for man, each with the corresponding disease that it causes. 

Reading. — 

Lesson 31. — Material Required. — If a case of amebic dysentery is 
available fresh fecal material, kept at body temperature, should be 
supplied — a small portion for every six men. If such material is not 
available, closely related organisms may be secured from the following 
sources : (a) Procure some of the ooze from the bottom of a pond or 
slowly moving stream ; place it in wide-mouthed bottles ; cover with a 
fair amount of water; add a few cubic centimeters of plain broth and 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 67 

allow the mixture to stand for four or five days, at room tempera- 
ture. Amebse will then be found in abundance in the scum that has 
formed at the surface of the water, and can be cultured on Musgrave 
and Clegg's agar, (b) Have available a number of mice — two for 
every six men — killed with chloroform, tacked up on boards and 
opened up; in the head of the cecum and the upper portion of the 
colon of every one out of two animals Entameba muris which closely 
resembles Entameba coli may be found in small numbers, (c) Have 
ready a number of frogs, pithed and tacked up ; in the intestinal con- 
tents the Entameba ranarum may be found and serve to give an 
idea of the morphology of the Entameba histolytica, {d) Have 
available also preserved human feces, containing the Endameba 
histolytica and Entameba coli in the encysted state; use 10 per cent, 
formalin in saline as preservative, (e) Supply also stained smears 
of material containing the two types of amebse. The specimens 
should be wet — fixed in alcoholic bichloride and stained with Dela- 
field's hematoxylin or Heidenhain or Rosenbusch's iron hematoxylin. 

Directions to the Student. — (1) If fresh human feces containing 
parasitic protozoa are available mount a small drop, emulsified 
with a little saline if necessary; cover with a cover-glass; ring with 
vaselin and examine first with the low power to locate the organisms, 
then with the middle power to study their structure and mode of 
locomotion. In an amebic dysentery stool the Entameba histo- 
lytica will be observed undergoing most active ameboid movements, 
during which the division of the cytoplasm into a hyaline ectosarc 
and a granular endosarc can readily be made out. Most of the 
organisms will be found to carry red cells in their interior. In such 
stools epithelial cells and bacteria, contrary to what one finds in 
bacillary dysentery, are not numerous. Do not confound swollen 
tissue cells containing a degenerating nucleus with quiescent 
amebse ; if in doubt, warm the slide by laying a heated coin on the 
slide and do not call the cell an ameba unless you can bring it to put 
out pseudopodia. 

Make careful drawings of moving and quiescent organisms ; show 
the small indistinct ring nucleus, the presence of food vacuoles, con- 
taining red cells; the granular endosarc and the hyaline ectosarc in 
quiescent forms, and the manner in which the pseudopodia are 
projected. (2) Mount a drop of preserved human fecal material 
containing encysted amebse both of the histolytica and the coli 
variety; note the large size of the latter and the presence of eight 
nuclei as compared with the four nuclei and the smaller size of the 



68 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



former. In the histolytica cysts from one to three chromidia can 
usually be made out, while the cysts of Entameba coli rarely con- 
tain chromidia; when present they appear like threads and spicules. 
Draw. (3) If fresh human material is not available, mount a drop 
of the surface scum from the culture of the free living type that was 
supplied to your group; cover with a cover-glass; ring with vaseline 
and examine as above. Make drawings. (4) Mount a droplet of 
the contents of a mouse's colon; dilute with a droplet of saline and 
search for amebse. The Entameba muris closely resembles the 
Entameba coli, and its cysts, which may be found in the feces, if 
the vegetative form occurs in the intestine, like those of the Enta- 
meba coli contain eight nuclei. Entameba coli is not as actively 
motile as Entameba histolytica; its pseudopodia are broader and 
there is no separation of the cytoplasm into ecto- and endosarcs 
in the quiescent organism. The few vacuoles contain bacteria but 
no red cells, and the nucleus is quite distinct. Draw. (5) Mount 
a drop of the intestinal contents of a frog and search for amebse 
resembling the Entameba histolytica. Study their structure and 
locomotion. Draw. (6) Study the stained preparations and make 
drawings. 

Answer the Following Questions in Writing. — What organism is 
the cause of chronic dysentery ? Who first established the causative 
role of the organism in question in reference to chronic dysentery 
and liver abscess? Is the Entameba coli pathogenic? Prepare a 
table showing the points of difference between the histolytica and 
the coli (a) in the motile, (6) in the encysted stage. To what class 
of protozoa do the amebse belong? To what subclass? To what 
order? 

Reading. — 

Lesson 32. — Material Required. — Furnish every six students with 
a small sample of a freshly voided stool that has been kept at body 
temperature containing (a) Trichomonas intestinalis, (b) Lamblia 
intestinalis, (c) Balantidium coli. If fresh material is not available 
furnish specimens that have been preserved with formalin. If such 
material also is not at hand, closely related forms may be procured 
from the following sources: (a) Trichomonads and Lamblias are 
common in the fresh feces of guinea-pigs, rats and mice; the fecal 
matter is conveniently emulsified in a little saline. (6) Opalina and 
Nyctotherus cordiformis may be found in the contents of the large 
intestine of frogs; Nyctotherus ovalis is frequently encountered in 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 69 



the intestine of the cockroach, (c) Paramecium may be obtained 
from amebic cultures in stagnant water. All such material can be 
readily preserved with formalin, (d) Furnish stained specimens 
of these various types. 

Directions to the Student. — Mount drops of the various materials, 
emulsified in a little saline if necessary; adjust cover-glasses and 
ring with vaselin. Examine all the specimens both with the low and 
the middle power. In the specimens containing trichomonads note 
their size and motility; the organism is pear-shaped; three flagella 
are directed forward; the fourth is directed posterially and clinging 
closely to the body of the organism forms an undulating membrane, 
which extends to the posterior pole. In the stained preparation 
note the nucleus. Draw these features. In some specimens of lam- 
bliasis cysts only are encountered; these are formed in the large 
intestine through the union of two individuals. They are oval, 
the longer diameter measuring about one and a half of a red cell. 
The organism proper is pear-shaped and provided with four pairs of 
flagella; the posterior surface is convex; the anterior surface presents 
a depression in which the mouth opening of the organism — the 
peristome — is located. In the Balantidium specimen note the large 
size of the organism which is covered with actively motile cilia which 
are arranged in longitudinal rows; they are grouped most densely 
about the funnel-shaped mouth. In the posterior portion are two 
contractile vacuoles which communicate with each other and open 
to the outside — the anus. In the stained specimen note the two 
nuclei, of which the macronucleus is kidney shaped. Draw. Opa- 
lina, Nyctotherus and Paramecium are closely related to Balanti- 
dium. Opalina ranarum is oval in shape and flattened; it has no 
cystostome ; it contains numerous vesicular nuclei, but no contractile 
vacuoles; waste material is eliminated through a system of canals. 
Draw. Paramecium is a long, spindle-shaped organism, provided 
with a slit like cystostome on one side; it contains a macro- and a 
micronucleus, and two contractile vacuoles with excretory canals 
arranged about them in a rosette form. Draw. Nyctotherus cordi- 
formis' is kidney-shaped; its body is evenly covered with cilia in 
long rows; on one side of the long cystostome the cilia are longer 
and thicker than on the other. There is a kidney-shaped macro- 
nucleus with a micronucleus, lying in its concavity. In the posterior 
end there is a large vacuole which opens to the outside. Draw. 

Answer the Following Questions in Writing. — To what class, sub- 
class and order does each one of the following protozoa belong: 
8 



70 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



Trichomonas intestinalis, Lamblia intestinalis, Balantidium coli? 
To what class does Opalina, Paramecium and Nyctotherus belong? 
What is the pathological significance of each? What is the blood 
picture in uncomplicated cases of amebiasis (histolytica) ? What is 
it in cases complicated with liver abscess? 
Reading. — 

Lesson 33. — Material Required. — Furnish every group of six men 
with small samples of fecal material, properly emulsified with saline, 
either fresh or preserved with formalin (10 per cent.), containing 
the ova of (a) Tenia solium; (b) Tenia saginata; (c) Hymenolepis 
nana; (d) Dibothriocephalus latus; (e) Fasciola hepatica; (/) 
Clonorchis endemicus; (g) Paragonimus westermani; (h) Schisto- 
somum hematobium; (i) Schistosomum japonicum. 

Directions to the Student. — Mount a drop of each one of the fecal 
specimens furnished your group; adjust a cover-glass and examine, 
first with the low power of the microscope, so as to learn to rapidly 
locate the ova and to form a mental picture of their size, form, 
color and general appearance. Then examine each specimen with 
the middle power and make careful drawings. 

In the tape-worm specimens note that the ova are sometimes sur- 
rounded with an oval vitelline membrane containing yolk spheres. 

The eggs of Tenia solium and Tenia saginata are round, of a 
brownish color and surrounded by a thick, radially striated mem- 
brane. In the interior the six hooklets of the embryo-hexacanth or 
oncosphere — can usually be made out. The ova of Hymenolepis 
nana are colorless and present two distinct membranes; the inner 
one has two knobs from which filaments emanate; in the interior 
the hooklets of the embryo can be made out. The eggs of D. latus 
are oval; they are enclosed in a brown envelope which is operculated 
— a little pressure on the cover-glass will cause the operculum to 
open. The contents consist of protoplasmic spherules, all of about 
the same size, which are lighter in color at the center than at the 
periphery. The embryo, which at times escapes from fully developed 
ova, on pressure, is ciliated and contains six hooklets. The eggs of 
Fasciola hepatica, Paragonimus westermani and Clonorchis endemi- 
cus are brown, oval and operculated; those of Clonorchis are 
enclosed in a colorless envelope; slight pressure upon the cover- 
glass will cause the little lid to open. The ova of Schistosomum 
hematobium are fusiform, yellowish, non-operculated and provided 
with either a terminal or a lateral spine; their shell is very thin. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 71 



The ova of S. japonicum are oval, transparent, but spineless, and 
in size and general appearance resemble those of the hookworm. 
When water is added to the fresh specimen the ciliated embryo, 
which can be made out in the transparent shell, escapes in about 
ten minutes and swims about quite actively. 

Answer the Follotuing Questions in Writing. — To what phylum, 
class and order does each one of the parasitic worms, whose ova you 
have just studied, belong? Name the intermediary host of each, 
through which infection of the human being occurs. Name the 
human habitat of the adult organism in each. 

Reading. — 

Lesson 34. — Material Required. — Furnish every student with a 
permanently mounted specimen of a mature segment of T. saginata, 
and every six to eight students with mounted specimens (a) of a 
mature segment of Tenia solium and Dibothriocephalus latus; (b) 
of heads and upper segments of the three tape-worms in question; 
(c) with entire specimens of Hymenolepis nana and with as many of 
the various flukes, whose ova were studied in the preceding lesson, 
as may be available. The gross specimens should be cleared in 
lactophenol and mounted in glycerin jelly. 

Lactophenol is composed of 1 part of pure phenol crystals, c. p., 
1 part of lactic acid, 2 parts of glycerin and 1 part of distilled water. 

To prepare glycerin jelly take 7 parts of gelatin, allow to soak in 
42 parts of distilled water for two hours; add 50 parts of glycerin; 
heat to boiling on a water-bath while stirring; add 1 part of phenol 
and finally filter through glass wool. 

To mount a specimen warm the jelly until it becomes fluid ; place 
the specimen in a small quantity of the jelly, adjust a cover-glass 
and after the jelly has coagulated ring with Kroenig's wax. This is 
prepared as follows: Melt 2 parts of bee's wax in an evaporating 
dish; slowly add 7 to 9 parts of powdered colophonium (rosin) 
while stirring and pour into small tin boxes. To ring a specimen 
heat a piece of wire, bent at a right angle ; dip it into the wax and 
seal the cover-glass along its edge. 

Furnish every six men also with small fecal samples — preserved 
with 10 per cent, formalin in saline, containing the ova of the 
following parasites: Ascaris lumbricoides, Oxyuris vermicularis, 
Necator americanus and Trichocephalus trichiurus. 

Directions to the Student. — (1) Make a careful drawing of a mature 
segment of T. saginata. Note the position of the genital pore leading 



72 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

into the genital sinus, and the vagina running to the uterus which 
presents from fifteen to thirty slender dichotomous branches; 
observe also the vas deferens and the testicles scattered through- 
out the proglottis. The genital pores in T. saginata occur on 
alternate sides in the different segments. (2) Next, draw a 
mature segment of T. solium and note the difference in the 
number and arrangement of the uterine ramifications. (3) 
W ith a hand magnifying lens, or the ocular of your microscope 
inverted, study the difference in the structure of the head of 
T. solium as compared with T. saginata. Note the general form, 
the presence or absence of a rostrum, the presence or absence 
of hooklets and the appearance of the sucking discs. Draw. (4) 
Study and draw the structure of a segment of Dibothriocephalus 
latus along the same lines as in the case of T. saginata. The mature 
segments are broader than long; the genital pores are in the median 
line. (5) Study and draw the mounted specimen of Hymenolepis 
nana; examine the head and note the presence *of a retractile 
rostrum surmounted by a circle of hooklets. The genital pores are 
non-alternating. (6) Make a careful drawing of an adult Fasciola 
hepatica. Note its leaf-like form, the presence of two suckers — one 
located on the ventral surface. Between the two is the geuital pore 
which leads into a skein-shaped uterus; note also the greatly 
branched testicles. Draw. (7) Mount drops of the fecal specimens 
furnished your group; adjust cover-glasses and examine, first, with 
your low power and then with your middle power for the presence 
of the corresponding ova; note their size, color, form and structure 
and make careful drawings of each type. Look for the presence of 
Charcot-Leyden crystals, (a) The fertilized ova of Ascaris lumbri- 
coides are elliptical and provided with a thick, transparent shell; 
there is also an external coating which is usually colored a yellowish 
brown and provided with protuberances giving the entire egg the 
appearance of an English walnut when viewed with a certain focus. 
The ovum proper is single-celled. When freshly passed the eggs are 
colorless. Unfertilized eggs are also frequently seen; these are 
long and more opaque than the fertilized ones. Sometimes ova are 
met with which have lost their albuminous outer coating and present 
a smooth, colorless shell — these are often not recognized as Ascaris 
eggs by the beginner, (b) The ova of Oxyuris are colorless, flattened 
on one side and provided with a smooth double-contoured shell; 
they contain an embryo at the time that they are passed in the feces, 
(c) The ova of Necator have an oval form and a smooth surface; 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 73 



the shell has a double contour; the contents are granular, grayish in 
color and on careful observation will be seen to be composed of a 
varying number of segments; in material that has stood for twenty- 
four to forty-eight hours motile embryos may be seen within the 
shell, (d) The ova of Trichocephalus trichiurus are elliptical in 
shape, brown in color and provided with a double-contoured shell, 
with a depression at each end, which is closed by a little knob-like 
lid. The contents are coarsely granular and contain a single cell 
at the time that the eggs are passed with the feces. 

Answer the Following Questions in Writing. — Name the class and 
order to which each one of the parasitic worms belong whose ova 
you have just studied. How does infection with each occur in man? 
Name the habitat of each in man. What is the most constant 
feature of the blood picture in the intestinal helminthiases? Where 
and under what pathological conditions may Charcot-Leyden crys- 
tals be encountered aside from the helminthiases? What is meant 
by a miracidium? What are cercarise? What symptoms on the part 
of the patient would lead you to examine the stool for intestinal 
parasites or their ova? 

Reading. — 

Lesson 35. — Material Required. — 1. Furnish every group of six 
to eight students with as many gross specimens of the following 
adult parasites as maybe available: (a) Ascaris lumbricoides — male 
and female; (b) Necator americanus — male and female; (c) Tricho- 
cephalus trichiurus — male and female; (d) Oxyuris vermicularis — 
male and female; (e) Trichinella spiralis — male and female; (/) 
Strongyloides intestinalis — female. 

2. Furnish also (a) preserved tissue specimens — taken from a dog 
showing hookworms attached to the intestinal mucosa; (b) meat 
infected with trichinella — trichinous rats; (c) sections of muscle 
tissue showing encysted trichinella embryos; (d) fecal material 
containing the embryonic worms of Strongyloides intestinalis — if 
possible living. 

Difections to the Student. — (la). In examining the specimens of 
Ascaris use a hand lens or the ocular of your microscope inverted. 
Note the three oral papillae, one dorsal and two ventral; the trans- 
verse striation and color of the body; the tail end of the male is 
rolled up on its ventral surface and provided with two spicules which 
project from the subterminal cloaca. The genital aperture of the 
female is located directly behind the anterior third of the body. 



74 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

Draw, (lb) Examine the hookworms with the low power of the 
microscope. Note their size. At the opening of the buccal cavity 
of Necator americanus there will be seen two broad lips and a blunt 
dorsal conical tooth; beside these there are four buccal lancets. At 
the tail end of the male will be seen the umbrella-shaped copulatory 
bursa; the tail end of the female is pointed; the genital aperture 
of the female is located in the upper half. Draw, (lc) Examine the 
Trichocephalus specimen with a hand lens and the low power. Note 
the whip-like anterior end of the worm. The tail end of the male is 
curled in a spiral and presents a single terminal spicule. The female 
is a little larger than the male; its tail end is not curled upon itself. 
Draw. (Id) Examine the Oxyuris specimens with the low power; 
the female is the longer; the tail end of the male is bent upon itself 
and provided with a single spicule; that of the female is straight. 
In both the bulbous esophagus can be made out. The female is 
often distended with ova. Draw, (le) Examine the Trichinella 
specimens with the low power; the female is longer than the male. 
The esophagus appears as a serrated line. The female has a rounded 
posterior extremity provided with a slit-like cloaca. The enlarge- 
ment in the posterior end of the male is the testicle; the male has 
two tongue-like appendages without a spicule. Draw. (1/) 
Examine the strongyloides specimens with the low power. All the 
adult worms that are found in the intestinal mucosa are female. 
Note the pointed, four-lipped mouth and the filariform esophagus. 
The tail end is sharply pointed; the genital opening is located near 
the beginning of the lower third of the body. In the uterus a row of 
ova may be seen which closely resemble those of the hookworm. 
The embryos develop in situ and find their way into the intestine, 
where the ova are but rarely found — only after active purgation. 
Draw. (2a) Note the firm attachment of the hookworm to the 
mucosa. (26 and c) Examine the meat with a hand lens and note 
the lemon-shaped cysts. Examine the section with the low power; 
note that the long axis of the cyst is placed in the long axis of the 
muscle fibre. Each cyst contains one and sometimes two or more 
larvae. Note the large number of eosinophilic leukocytes about each 
cyst. Make careful drawings. (2d) Mount drops of the fecal 
material containing strongyloid embryos; cover with a cover-glass 
and search for motile larva?; note their method of progression. 
Examine one that has come to rest, with the middle power; note 
the pointed tail and the double esophageal bulb (rhabditiform 
larvae). Make drawings. 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 75 

Answer the Following Questions in Writing. — How soon after 
infection with trichinella would you expect to find the larvae in the 
blood ? How soon would you be apt to find them in the muscles ? 
"Which muscles are usually the most heavily infected? How does 
infection of the human being take place? What role does the rat 
play in the dissemination of the disease ? What organism is respon- 
sible for so-called Cochin-China diarrhea? In what parts of the 
United States is the organism also met with? 

Reading. — 

Lesson 36. — Material, Apparatus and Reagents Required. — Supply 
every six men with 600 c.c. of an albuminous urine — marked A — 
and 600 c.c. of a urine containing sugar, acetone and diacetic acid — 
marked B. The urine should have been preserved with chloroform. 
Furnish about 50 c.c. also of a urine containing bile pigment; mark 
this C — to be examined for bile pigment only. 

Of reagents furnish every six students with 6 oz. of concentrated 
nitric acid; 1 oz. of a saturated solution of sodium chloride; 1 oz. 
of 5 per cent, acetic acid; 1 oz. of a 10 per cent, solution of potassium 
f errocy anide ; 6 oz. of Fehling's solution No. 1 and 6 oz. of Fehling's 
solution No. 2; 6 oz. of Nylander's solution; half a cake of bakers' 
yeast; 1 oz. of tincture of iodin, diluted 1 to 10 with alcohol; a few 
grams of sodium nitroprusside ; 4 oz. of a 10 per cent, solution of 
ferric chloride; 6 oz. of 20 to 30 per cent, sodium hydrate solution; 
6 oz. of glacial acetic acid; 6 oz. of Obermeyer's reagent; 2 oz. of a 
5 per cent, solution of silver nitrate; 4 oz. of chloroform. 

Furnish every individual student with the following: 1, 2 or 3 oz. 
conical glass; a few pieces of filter paper and a funnel; a dozen 
test-tubes of ordinary size; a couple of agglutination tubes; a 
test-tube rack; a couple of 4 oz. beakers; a urinometer; a 100 c.c. 
cylinder; red and blue litmus paper and four agglutination tubes. 

Directions to the Student. — From the two samples of urine fur- 
nished your group take a specimen of 100 c.c. each in your two 
beakers and mark them A and B, to correspond to the stock samples. 
Examine each specimen according to the following plan: Note the 
color; is it light, medium or dark amber; or is it brownish or reddish 
in color; test the reaction with litmus paper; is it acid, alkaline or 
amphoteric. Take the specific gravity; do not drop the urinometer 
into the urine, but immerse it with care and see to it that it is dry 
before being used. 

(1) Next, test for albumin, using each one of the following three 



76 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



tests: (a) The cold nitric acid test. Place 10 to 15 c.c. of urine 
in the conical glass; incline the glass and allow concentrated 
nitric acid to flow down its side; being heavier than the urine it 
will sink below the latter; along the line of contact a white turbid 
ring will form, if albumin be present — either immediately or on 
standing for a few minutes; the density and the width of the ring 
will be proportionate to the amount of albumin present. On 
standing a white ring will also form higher up than the line of contact, 
in the clear urine; this is due to the precipitation of urates. (6) If 
albumin has been demonstrated with the above test, place 10 to 15 
c.c. (1 to 1J inches) of the same urine in a test-tube; add a few drops 
(3 to 10) of 5 per cent, acetic acid, so as to make the specimen 
markedly acid; then add approximately one-sixth of its volume 
of a saturated solution of sodium chloride and carefully bring to 
the boiling-point; if albumin is present a turbidity will result, and 
if the amount be larger than a trace, the substance will flake out 
in flocculent form, (c) Take another sample of the same urine; 
acidify strongly with acetic acid and add a few drops of 10 per 
cent, potassium ferrocyanide solution; if albumin is present a 
turbidity or a flocculent precipitate will develop, according to the 
amount present. 

(2) Now test for sugar, using each one of the following 
three tests: If albumin was present, acidify the urine with 
acetic acid, boil and filter. If no albumin was present, boil 
a little of the urine to remove traces of chloroform, which had 
been added as a preservative and which would interfere with 
the tests. Then proceed as follows: (a) Place 5 c.c. approxi- 
mately of Fehling's solution No. 1 in a test-tube; add an 
equal volume of Fehling's solution No. 2; mix well; dilute with 
about four volumes of water and bring the upper portion of the 
mixture to the boil, holding the tube with a manifolded strip of 
paper; now add about 1 c.c. of urine, warm the mixture for a moment 
and set the tube aside. In the presence of sugar a yellowish or red- 
dish precipitate will develop either at once or on standing for a few 
minutes. The development of a greenish color does not indicate 
sugar. When in doubt apply the fermentation test to the same 
sample (see below, c). 

(b) In lieu of the above test Nylander's test may be used. 
Place 5 to 10 c.c. of the urine in a test-tube; add one-tenth the 
amount of Nylander's solution and bring the mixture to a simmer; 
keep this up for a couple of minutes. In the presence of sugar a 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 77 



grayish, dark brown and finally a black precipitate is obtained; 
the rapidity with which the change in color appears, as well as its 
intensity, depends upon the quantity of sugar that is present. 

(c) Take a small bit of yeast; emulsify it in some of the 
urine to be examined; introduce one of the agglutination tubes, 
mouth downward into a large test-tube; fill the tube with the 
yeast-urine mixture; close it with your finger and invert it; slowly 
bring it back to its former position — the idea is to fill the agglutin- 
ation tube inside of the large tube; finally pour out enough of the 
urine until the top of the agglutination tube becomes visible; the 
latter must not contain any air bubble whatsoever. Fill another 
set of tubes with plain water, in which a little yeast has been emulsi- 
fied, and use this as a control. Place both in the incubator at 37° 
to 40° C, until the next day, and then ascertain whether any bubble 
of gas has appeared at the top of the agglutination tube; if so, a 
fermentable sugar must have been present. This test must always 
be employed when the two other tests described furnish a doubtful 
result. 

(3) Test for Indican as Folloivs. — To about 5 c.c. of urine add an 
equal amount of Obermeyer's reagent and 1 c.c. of chloroform; 
close the tube with your thumb and invert a dozen times. In the 
presence of a normal quantity of indican the chloroform will either 
show no color at all or a light blue. A dark blue indicates an 
increase. 

(4) Test for Diacetic Acid as Follows. — To about 5 c.c. of urine 
add 10 drops of a 10 per cent, chloride of iron solution. If a Bur- 
gundy color develops the reaction may be due either to diacetic 
acid or to the presence of a drug — salicylic acid or one of its deriv- 
atives. To differentiate between the two dilute 5 c.c. of urine with 
an equal volume of water, boil down to the 5 c.c. mark and then add 
the chloride of iron. If the color now does not appear the original 
reaction was due to diacetic acid. 

(5) Test for Acetone. — Take 5 c.c. or more of urine in a test-tube; 
add a few crystals of sodium nitroprusside ; shake until the latter 
has dissolved; alkalinize strongly with 20 to 30 per cent, sodium 
hydrate solution; then allow glacial acetic acid to slowly flow down 
the side of the tube. The appearance of a Burgundy color at the 
zone of contact indicates the presence of acetone. 

(6) Test for Chlorides. — If albumin is present this must first be 
removed by boiling, after acidifying with a few drops of 5 per cent, 
acetic acid; filter; to about 10 c.c. of the filtrate add approximately 

9 7 



78 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

1 c.c. of concentrated nitric acid and then a few cubic centimeters 
of a 5 per cent, solution of silver nitrate. If a heavy flocculent 
precipitate develops the chlorides are present in normal amount. 
If an opalescence only results they may be regarded as absent. 

(7) Test for Bile Pigment. — In the presence of bile pigment a 
greenish ring will be seen to develop at the zone of contact between 
the urine and the nitric acid when the latter test is applied, as in 
the examination for albumin. When in doubt overlay about 5 c.c. 
of urine with 2 c.c. of tincture of iodin that has been diluted 1 to 10 
with alcohol. At the zone of contact an emerald-green ring will 
develop. Apply this test also to urine C. 

Write up your findings in A, B, and C in the form of a formal 
report or in the form of a letter directed to a fellow doctor. 

Answer the Following Questions in Writing. — What does the 
specific gravity of the urine indicate? What is the normal volume 
for twenty-four hours? What is meant (a) by oliguria, (6) anuria, 
.(c) polyuria? What does a low specific gravity indicate? Under 
what pathological conditions would you expect a low specific gravity 
and under what conditions a high one? What would a greenish- 
brownish color of the urine suggest? What a red or reddish-brownish 
color? Under what conditions would the reaction of the urine be 
alkaline? What would an alkalinity due to volatile alkali indicate? 
How would you determine whether the alkalinity in a given case 
was due to fixed or volatile alkali? Under what pathological con- 
ditions are the chlorides absent? Under what conditions would bile 
appear in the urine? Under what conditions would you expect 
acetone and diacetic acid in the urine? What are the albumins 
that are ordinarily met with in the urine? What is meant by 
Bence- Jones albumin? Enumerate the conditions under which 
albumin may appear in the urine? What is meant by alimentary 
glycosuria? How would you differentiate between the latter and 
true diabetes — arrange your statements in tabulary form? What 
does an increase in the amount of indican indicate? 

Reading. — 

Lesson 37. — Material, Apparatus and Reagents Required. — Fur- 
nish every six students with 150 c.c. each of a normal, of an albu- 
minous and of a diabetic urine— marked correspondingly — ; note the 
specific gravity and the twenty-four hour amount of each on the 
beakers. All three should have been preserved with chloroform. Also 
supply every six men with 150 c.c. of Esbach's reagent; 500 c.c. of 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



79 



Benedict's solution for the quantitative estimation of sugar; 200 
grams of crystallized sodium carbonate and a few grams of talcum 
powder; 1 oz. of a saturated solution of potassium chromate and 300 
c.c. of a -f^- solution of silver nitrate; 1 oz. of 5 per cent, acetic acid; 
1000 c.c. of distilled water. 

Supply every man individually with the following apparatus: 
One 100 c.c. cylinder; one 150 c.c. beaker; one urinometer; one 50 
c.c. Mohr's burette, graduated in tenths, with stand; one evaporat- 
ing dish — 150 c.c. capacity; one stirring rod with rubber tip; one 
tripod with wire gauze; one 5 c.c. pipette; one 10 c.c. pipette; one 
25 c.c. pipette; one Esbach albuminimeter with stopper and stand. 

Directions to the Student. — (A) Fill your albuminimeter with the 
albuminous urine to the mark U — lower meniscus at U — ; add 
Esbach's reagent to R — lower meniscus; close the tube with its 
stopper; invert it a number of times; then set it aside in its stand 
— vertically — for twenty-four hours. (B) Determine the quantity 
of sugar present in the diabetic urine as follows: If the specific 
gravity of the urine is 1030, dilute five times; if it is higher, dilute 
ten times, i. e., 1 in 5 or 1 in 10. Calculate out the requisite amount 
of urine to make up 50 c.c. of its dilution. 1 Use the 10 c.c. pipette 
and dilute with water in the 100 c.c. cylinder. Fill your burette 
with the diluted urine; see to it that there are no bubbles in the 
rubber cuff and that it is full to the tip. Place 25 c.c. of Benedict's 
solution into your evaporating dish, together with 10 to 20 grams 
of sodium carbonate — roughly half a teaspoonful — and a pinch of 
talcum powder; arrange the evaporating dish on the tripod, under- 
neath the tip of the burette which has been charged with the urine, 
as just directed. Note your reading and then slowly heat the mixture 
in the evaporating dish to the boiling-point. While ihe solution is 
gently boiling, run in the urine, rather rapidly, stirring all the time, 
until every trace of blue has disappeared. ^Yater may be added 
during the process, if the fluid becomes too concentrated. Take 
your second reading and ascertain the number of cubic centimeters 
of the diluted urine that were necessary to bring about decoloriza- 
tion ofthe Benedict solution. Make a second titration and average 
your results. The titer of the Benedict solution is such that 25 c.c. 
will be reduced by 0.05 gram of dextrose. Calculate the percentage 
present in the urine under examination, and from this the total 

1 Before diluting the urioe, boil it in a beaker for a few moments to remove traces 
of chloroform, which was used as a preservative and which itself reduces cupric 
salts. 



80 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

amount voided in twenty-four hours. (C) Determine the quantity 
of chlorides present in the normal specimen of urine as follows: 
Charge your burette, after carefully washing it out, first with tap 
and then with distilled water, with from 20 to 30 c.c. of silver 
nitrate solution — fill to the tip and eliminate air bubbles from cuff. 
With a pipette measure off 5 c.c. of the urine; place this in a beaker; 
add 50 to 75 c.c. of distilled water and 10 to 15 drops of the solution 
of potassium chromate as indicator; take your reading and, while 
stirring, allow the silver nitrate solution to run into the diluted urine 
until a reddish color appears and persists; then take your second 
reading; the difference represents the number of cubic centimeters 
of the silver solution which were necessary to precipitate the chlor- 
ides contained in 5 c.c. of urine; 1 c.c. of the silver solution will pre- 
cipitate the chlorine contained in 0.00585 gram of sodium chloride. 
Calculate the percentage of NaCl contained in the urine examined 
as well as the amount eliminated in twenty-four hours. (D) Deter- 
mine the chlorides present in the albuminous urine. To this end 
take about 10 c.c. of urine; acidify with 5 to 10 drops of 5 per cent, 
acetic acid; boil and filter — to remove the albumin; then proceed 
with the filtrate as directed sub. C. Submit all your quantitative 
results in the form of a letter, addressed to a colleague. Give the 
total quantity of urine, specific gravity, percentage and total amount 
of albumin, sugar and chlorides voided in twenty-four hours. 

Answer the Following Questions in Writing. — What is the normal 
output of chlorides, of total nitrogen, of urea, of uric acid and of 
creatinin in twenty-four hours? What is the normal two-hour 
response to Mosenthal's nephritic test-meal (a) as regards the 
quantity of urine voided; (b) as regards the specific gravity; (c) as 
regards the elimination of sodium chloride, and (d) as regards the 
elimination of the total nitrogen? Give findings in tabulary form. 
In what respect does the response of the nephritic differ from that 
of the normal individual? What is meant by the functional renal 
tests? How is Ambard's coefficient obtained? What information 
may be derived from the lactose test and how is the latter conducted? 

Reading. — 

Lesson 38. — Material, Apparatus and Reagents Required.— (A) 
For the Performance of the Permeation Test. — Administer the phenol- 
sulphonephthalein test to a number of normal and nephritic indi- 
viduals and have the urine collected from each, at the end of one 
hour, and after two hours, following the injection of the drug; pre- 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 81 



serve the specimens with chloroform, if the test is given the day 
before the exercise, and mark the bottles with the name of the 
patient as well as "after one hour" and "after two hours." Furnish 
every six men with a normal set and one from a case of nephritis. 

Supply every six men with the following: one 250 c.c. graduate; 
100 c.c. of a 10 per cent, solution of sodium hydroxide; one liter of 
distilled water. Have available as many Sahli colorimetric tubes 
and stands with the necessary standard color tubes (I, II and III) 
of phenolsulphonephthalein (as furnished by E. Leitz & Co.) as 
possible, together with a corresponding number of test-tubes, 
marked at 2 c.c, 5 c.c, and 10 c.c. respectively; a 2 c.c pipette and 
an ordinary dropping pipette provided with a rubber nipple. 

Directions to the Student. — Let one man of each group act as 
" preparateur" for his group. His duty shall be to measure the quan- 
tity of urine voided by each case under examination, taking the one- 
hour and two-hour specimen of each separately. He shall note the 
quantity on each bottle or glass, and shall then add 10 c.c. of a 10 
per cent, sodium hydroxide solution, and dilute each specimen with 
water up to the 200 c.c. mark. Note that the color of the urine turns 
reddish. 

Every student of the group is then to withdraw 2 c.c of this 
colored fluid, which is received in the graduated test-tube and 
examined as follows: dilute with distilled water to the 10 c.c. mark; 
mix well. Transfer enough of this diluted fluid to the colorimetric 
tube to reach the mark 50. Now compare the color with the stand- 
ard I and II. If it is darker than I it is diluted, drop by drop, with 
water until the colors match, the percentage being read off the same 
as in estimating the hemoglobin. If tube II is used the result must be 
divided by 2. Should the color be too light after the urine has been 
diluted to 10 c.c. prepare another specimen and dilute only to 5 c.c. 
Examine this as just directed, but divide the final result by 4. If 
the urine contains very small amounts of the excreted phenol- 
sulphonephthalein, then after it has been made up to 200 c.c. it 
should be poured directly into the calibrated tube up to the mark 50. 
The color must then be compared with the special color tube III, 
in which the yellowish tint of the less diluted urine has been com- 
pensated. Water is now added, until the colors match, when the 
reading obtained is divided by 20. Write up your findings in the 
form of a report addressed to a colleague. 

Answer the Following Questions in Writing. — How soon after the 
injection should the phenolsulphonephthalein appear in the urine 



82 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



under normal conditions? How large a percentage of the injected 
material should be eliminated during the first hour and how large 
a percentage during the second hour? What would your findings 
be if the integrity of the kidneys were seriously impaired? What 
are the indications for the use of the test? 
Reading. — 

(B) The study of acidosis by the determination of the tension of the 
alveolar carbon dioxide. Furnish every six men with a Marriott 
outfit for the determination of the alveolar carbon dioxide tension; 
have available for every individual man a calcium chloride tube or 
similar contrivance, moderately tightly packed with cotton (discard 
the cotton after use and sterilize the tube by boiling or in the hot- 
air sterilizer). 

Two students should work together, one as examiner, the other 
as subject. 

First go over the contents of the box; then proceed as follows: 
By means of an atomizer bulb fill the rubber bag of the outfit a 
little less than half-full with air, and clamp off the rubber tube. 
Interpose a fresh cotton filter tube between the bag and the mouth- 
piece. The subject, at the end of an expiration, takes the mouth- 
piece into his mouth while the examiner holds the subject's nose 
closed; the individual now breathes back and forth into the bag, 
four times in twenty seconds, the examiner marking the time. The 
tube is then immediately clamped off and the analysis of the respired 
air started within three minutes, as carbon dioxide escapes through 
rubber. To this end the test-tube accompanying the instrument is 
charged with 2 or 3 c.c. of the accompanying solution of bicarbonate 
of soda, colored with phenolsulphonephthalein, when air from the 
bag is forced through (by means of the finely drawn out glass tube), 
until no further change in color takes place. The tube is then 
stoppered and the color compared with the standards in the little 
stand. The number on the standard tube which is matched indi- 
cates the corresponding tension. Make duplicate determinations 
until two successive ones do not differ by more than 2 mm. 

Answer the Following Questions in Writing. — What is meant by 
acidosis? Under what pathological conditions is it encountered? 
Name the acids which are concerned in its production? In what 
manner does their production lead to deleterious results? What is 
meant by the reserve alkali of the blood? What is the normal hydro- 
gen ion concentration of the blood? W T hat is found in acidosis? 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 83 



What causes this change ? Why may the carbon dioxide tension of 
the alveolar air serve as an index of the existence of acidosis and 
its extent? What is the normal tension? In what direction is it 
changed in acidosis? What is meant by the ammonia coefficient 
of the urine? What is it normally? In what direction does it tend 
in acidosis? Why may the ammonia coefficient serve as an index 
of the existence of acidosis and its degree? What is meant by alka- 
linosis? Under what condition does it occur? What is the mother 
substance of acetone in cases of acidosis? Write the chemical for- 
mula expressing its origin. 
Reading. — 

Lesson 39. — Material Required. — Have available for every six 
students a set of tubes containing as extensive a collection of urinary 
sediments or centrifugates as possible, each being suspended in a 
few cubic centimeters of urine or saline. It is desirable to collect 
large amounts of urine of each type and to preserve it with chloral. 
The sediment may then be allowed to form spontaneously; the 
supernatant fluid is pipetted or siphoned off, when the remaining 
sediment may be further concentrated as desired and finally distrib- 
uted in tubes — one tube of each kind for every six men. Provide 
each tube with an ordinary pipette and label it as to contents. One 
set of tubes (A) should contain those non-organized constituents 
which might be encountered in an acid urine, such as (a) uric acid — 
various forms; (b) calcium oxalate — various forms; (c) primary 
calcium phosphate and (d) cystin. One tube (e) should contain 
those non-organized constituents which one would meet with in an 
alkaline urine — the alkalinity being due to fixed alkali — viz., basic 
phosphate of magnesium and calcium, ammonio-magnesium phos- 
phate, calcium carbonate and neutral calcium phosphate. Another 
tube (J) should contain those non-organized constituents which we 
would meet with in an alkaline urine whose alkalinity is due to 
volatile alkali, viz., ammonium urate — various forms — in addition 
to the constituents of the preceding tube. (B) The next set of speci- 
mens should contain (a) mucous cylinders, (6) the various kinds of 
casts, (c) epithelial cells, (d) pus corpuscles, (e) red cells and red cell 
shadows (from a case of nephrolithiasis) and (/) spermatozoa. (C) 
A final tube should contain the concentrated morphological con- 
stituents of a case of renal tuberculosis, including the bacilli in 
question. 



84 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 



Reagents Required. — For every six men 1 oz. of a 1 per cent, 
aqueous solution of eosin; 4 oz. of Gabbett's carbol fuchsin solu- 
tion; 4 oz. of Gabbett's acid methylene-blue solution; 1 oz. 
of a 10 per cent, acetic acid solution; 1 oz. of 30 per cent, hydro- 
chloric acid; 1 oz. of 25 per cent, caustic soda solution; 1 oz. of 
ammonium hydroxide. Have available also staining trays, wash 
bottles and medicine droppers or capillary pipettes. 

Directions to the Student. — Mount drops of the various sediments 
on slides, spread the material out and adjust a cover-glass over a 
portion of each specimen. Examine first with the low power — with 
the condenser thrown out, and using the flat mirror, so as to secure 
a subdued light; then with the middle power, making use of that 
portion of the smear that has been covered with the cover-glass — 
more light is then needed, to which end the convex mirror is sub- 
stituted for the flat mirror. Now make careful drawings of your 
findings and append corresponding legends. Sub A-a, note that all 
uric acid crystals are colored — unless they are so thin that the quan- 
tity of coloring matter present is insufficient to be recognized by the 
eye. The key form is the rhomb or whetstone. Draw individual 
crystals and a group — color them with your pencil. Add a drop of 
25 per cent, caustic soda — do the crystals dissolve? Sub A-b, note 
that the typical oxalate crystal, no matter how small, may always 
be recognized under the low power by the appearance of a dark 
surface side by side with a brightly illuminated surface — a black 
spot alongside of a high light. Examine with the middle power and 
note that this is due to the octahedral form of the crystals; note the 
highly refractive cross in the interior which comes out on focussing, 
and which in the larger specimens may even be seen with the low 
power. Allow a drop of acetic acid to flow under the cover-glass — 
the crystals do not dissolve; substitute a drop of hydrochloric acid — 
do they dissolve? Are the crystals colored? Sub A-c, note the 
prismatic form of the crystals; are they colored? Do they dissolve 
in acetic acid? Draw. Sub A-d, note the hexagonal form of the 
cystin platelets. Are they colored? are they soluble or insoluble in 
the following reagents — hydrochloric acid, acetic acid, ammonia? 
Draw. Sub A-e, note the acicular crystals of neutral calcium phos- 
phate, the occurrence of basic phosphate of calcium and magnesium 
in granular form or of the latter in the shape of large highly refrac- 
tive plates, and the large prismatic crystals of ammonio-magnesium 
phosphate (triple phosphate) in the shape of the lid of a German 
coffin (coffin-lid crystals). All these are soluble in acetic acid — test 



LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 85 



this. Draw. Sub A-f, note the dumb-bell shaped crystals of cal- 
cium carbonate, and the fact that they dissolve in acetic acid, with 
the evolution of gas bubbles. Draw. Ammonium urate occurs in 
the form of brownish spherical bodies of variable size, which are 
sometimes composed of delicate needles, while at others they are 
amorphous and sometimes beset with prismatic spicules — thorn- 
apple forms. Ammonium urate is soluble in acetic acid and hydro- 
chloric acid; subsequently rhombic crystals of uric acid separate 
out. Sub B-a, note the irregular, longitudinally striated, band-like 
structure of the mucous cylinders. Draw. Are they soluble in 
dilute acetic acid? Sub B-b, note the irregular, polyhedral or fusi- 
form and rolled-up appearance of the so-called vaginal epithelial 
cells and the large, flat, roughly quadrangular cells from the urethra. 
Observe the large round and oval, sharply contoured cells from the 
bladder. Draw. Sub B-c, note the parallel outline and solid, 
sausage-shaped appearance of the true casts. Note how essential 
it is to have a subdued light, by turning on full light, when it will 
be seen that the hyaline casts have become invisible. Study the 
character of the "impedimenta" which some of the casts carry, 
such as black amorphous granules (finely granular and coarsely 
granular casts), brown granules (brown granular casts), red cells 
(blood casts), leukocytes (leukocytic and pus casts — when leuko- 
cytes are abundant), renal epithelial cells (epithelial casts), fat 
globules (fatty casts). Draw all these appearances. Search for an 
epithelial cast; note the size and oval form of the cells, the granular 
character of the protoplasm and the presence of a single nucleus; 
next search for free cells of the same type. Draw. Add a drop or 
two of eosin solution to your specimen and note the manner in which 
the casts take the dye; the hyaline casts and the hyaline matrix of the 
compound hyaline casts is colored pink; the granules turn a deeper 
color. Search for large coarse casts presenting a vermilion color — 
many of these in the unstained specimen appear yellowish — waxy 
casts. Draw. Add a few drops of dilute acetic acid — what happens 
to the hyaline casts? Sub B-d, note the appearance of the leuko- 
cytes'—pus corpuscles — under the low power; add a drop of acetic 
acid, and examine with the middle power — the multiple nuclei are 
now well shown. Draw. Sub B-e, note particularly the blood 
shadows, i. e., red cells which have lost or are losing their hemo- 
globin. Learn to recognize the intact red cells with the low power, 
under which they appear as mere rings. Compare with the appear- 
ance of the leukocyte — the one a ring, the other a solid. Draw. 



86 LABORATORY SYLLABUS OF CLINICAL PATHOLOGY 

Sub B-f, note the appearance of the spermatozoa; learn to recognize 
them with the low power; search for corpora amylacea. Draw. 
Sub C, finally, prepare fairly thick smears of the centrifugate from 
the case of renal tuberculosis. Examine while wet ; note the presence 
of pus cells and red cells. Dry the specimen carefully by beating 
the flame of the Bunsen burner; then stain for tubercle bacilli, as 
directed in Lesson 19. Wash off ; dry and examine carefully with the 
oil-immersion lens; search for bacilli lying singly and in clusters. 
Make a composite drawing in colors. 

Answer the Following Questions in Writing. — What clinical signifi- 
cance attaches to the habitual passage of urine that shows a marked 
tendency to the spontaneous and abundant deposition of sediments 
composed of uric acid or oxalate of lime crystals? Does the appear- 
ance of an abundant sediment of crystals of a substance warrant 
the conclusion that the substance in question is being eliminated in 
increased amount? W T hat significance attaches to the appearance 
of hyaline tube casts in the urine? What conclusion would you draw 
from the appearance of blood casts and epithelial casts and leuko- 
cytic casts? What is pyuria? Enumerate the conditions under 
which pus might appear in the urine? How would you determine 
its source? Under what conditions does blood appear in the urine? 
What urinary findings would lead you to make an examination for 
tubercle bacilli? Should your search for the latter be negative and 
there be a clinical suspicion of the existence of renal tuberculosis 
nevertheless, what would your course of procedure be to prove its 
existence? What microorganism is most frequently associated 
with pyuria in women? How would you examine for its presence? 

Reading. — 



